Pairing processes for preparing alloreactive cytotoxic T cells

ABSTRACT

Provided in certain embodiments are methods for pairing patient cells and donor cells to prepare cytotoxic T cells. Such cytotoxic T cells could be administered to the patient for treating certain disorders, such as a cancer (for example, brain cancer).

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to provisional applications Ser.No. 61/229,229 filed on Jul. 28, 2009 and 61/229,233 filed on Jul. 28,2009, the entireties of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates, in one aspect, to a method of pairingpatient cells and donor cells to prepare cytotoxic T cells, which can beadministered to a patient for treating certain disorders, such as cancer(for example, brain cancer).

BACKGROUND OF THE INVENTION

T cells can be activated by an antigen presenting cell. An activated Tcell can bind to a cell that presents an antigen to which the T cell wasactivated via an interaction between a T cell receptor and majorhistocompatibility complex, and the activated T cell can kill the cellto which it is bound. It is possible to activate T cells from a donoragainst cells from a patient and generate cytotoxic T cells that killpatient cells. Such T cells are referred to as “alloreactive” T cells asthey are activated from donor cells and are active against the majorhistocompatibility complex (MHC) antigens (sometimes identified as humanleukocyte antigens or HLA) present on patient cells.

Alloreactive cytotoxic T cells can be prepared by isolating blood from apatient, separating white blood cells, and inactivating them. Theseinactivated patient cells can be mixed with white blood cells from adonor in a one-way mixed lymphocyte reaction. In the lymphocytereaction, T cells among the donor cell population are activated againstantigens presented by cells in the patient population, and activatedcytotoxic T cells are generated against the patient cells. The activatedcytotoxic T cells can be collected and administered to the patient.Cells in the patient, such as cancer cells, that display antigensrecognized by the cytotoxic T cells will be killed.

HLAMatchmaker (HLAMm) is an algorithm used in the transplantationsetting to predict alloantibody responses. HLAMm operates by findingpermissible mismatches between molecularly HLA-type donors andrecipients such to minimize rejection. When HLAMm is applied to thediverse HLA repertoire, it is able to predict B cell driven alloantibodygeneration following organ transplantation. However, the currentalgorithm has does not as reliably predict the T cell inducedgraft-versus host (GVH) disease.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a method of pairing apatient with one or more donors and of preparing alloreactive cytotoxicT cells, which can then be administered to a patient for treatingcertain disorders such as a cancer. One advantage of the presentinvention is to provide improved compositions and methods to prevent GVHdisease.

Methods described herein involve the identification of the presence orabsence of a partial mismatch between antigen information, orinformation determined from antigen information, from patient and donorcell pairs. Once a pairing is identified based on such a partialmismatch, alloreactive cytotoxic T cells can be developed, which, amongother things, have a stronger immunogenic activity than those producedfrom a pairing for which the partial mismatch is not identified. Thus, amethod according to the invention enables the identification of optimalpatient/donor matches for pairing cells from each and for preparingalloreactive cytotoxic T cells. These types of methods or processes arereferred to herein as “pairing processes.”

In one exemplary method according to the invention, patient cell anddonor cell pairs for the preparation of alloreactive cytotoxic T cellsare identified by first providing patient cell information, whichincludes patient cell antigen information determined at least partlythrough a high or intermediate resolution molecular sequencing method ofmajor histocompatibility complex (MHC) information. In other steps ofthis method, stimulator information from the patient cell antigeninformation is generated and compared to responder information generatedfrom donor cell antigen information, also determined at least partlythrough a high or intermediate resolution molecular sequencing method ofMHC information.

The presence or absence of a partial mismatch between the stimulatorinformation and the responder information is identified among patientcell and donor cell pairs, and a patient cell and donor cell match isselected for the preparation of alloreactive cytotoxic T cells based onthe presence of the partial mismatch. In another step of this exemplarymethod, cells of the patient are combined with cells of the donor in analloreactive cytotoxic T cell reaction based on the patient cell anddonor cell match. In different embodiments of this method, antigeninformation includes HLA class I antigen information and/or HLA class IIantigen information.

In some embodiments, patient or donor cell antigen information isdetermined at least partly through a high or intermediate resolutionmolecular DNA sequencing method. In other embodiments, patient or donorcell antigen information is determined at least partly through a high orintermediate resolution molecular RNA sequencing method. In still otherembodiments, patient or donor cell antigen information is determined atleast partly through a high or intermediate resolution molecular proteinsequencing method.

In different embodiments of the invention, the high or intermediateresolution molecular sequencing method includes one or more of asequence based typing (SBT) method, a sequence specific primer (SSP)method, a restriction fragment length polymorphism (RFLP) method, orsequence specific oligonucleotide (SSO) or restriction fragment lengthpolymorphism (RFLP) method.

In certain embodiments, T cell receptor interaction information includeseplet information. For example, partial mismatches may be determined bythe number or types of mismatched eplets.

The patient cell antigen information may be derived from one or morecell types, for example, from monocytes, antigen presenting cells,dendritic cells, lymphocytes, lymphoblasts, T cells, or the patient'stumor cells.

The presence or absence of a partial mismatch may be identified by usinga computer algorithm. Such algorithm may be configured to providestructurally based HLA matching and may include a string matchingalgorithm. This computer algorithm may be trained using a training setand may perform a statistical analysis on the training set, for example,a log-rank test.

An exemplary method according to the invention may also include the stepof exposing the cells of the patient to conditions that generateinactivated patient cells. Those conditions may include radiation andmitomycin C. The step of detecting the presence or absence of cytotoxicT cell activation may also be included in such exemplary method, forexample, by identifying the presence or absence of an activated T cellmarker or of interferon gamma. Certain embodiments include also the stepof determining the ratio of T helper 1 to T helper 2 cytokines.

In another aspect, the invention provides a method of treating a patientthat has a cancer, for example, a brain tumor such as a glioma. Thetumor can be primary or metastatic.

In an exemplary method according to the invention, cells that have beensubjected to an activation reaction may be administered to animmuno-privileged site of the patient such as the brain. In certainembodiments, the cytotoxic T cells are purified prior to beingadministered to the patient.

An exemplary method according to the invention may further include thestep of detecting the presence or absence of cytotoxic T cell activityin the patient. For example, the presence or absence of cancer reductionin the patient may be evaluated. In some embodiments of the invention,the cytotoxic T cell activation and/or the cytotoxic T cell activity maybe compiled in a training set for the algorithm.

Additional aspects and embodiments of the invention can be found in thefollowing description, examples, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings illustrate embodiments of the invention and are notlimiting. For clarity and ease of illustration, the drawings are notmade to scale and, in some instances, various aspects may be shownexaggerated or enlarged to facilitate an understanding of particularembodiments.

FIG. 1 shows Human Leukocyte Antigen (HLA) types of responding braintumor patients (BTP) and alloreactive cytotoxic T lymphocytes (alloCTL)donors:eplet number/type mismatch assessed by an algorithm.

FIG. 2 shows a flowchart of an embodiment of a method described herein.

FIG. 3 shows a flowchart of an exemplary application of an algorithm tothe method of FIG. 2.

FIG. 4 shows an exemplary computing environment where data processingand the algorithm of FIG. 3 may be utilized.

FIG. 5 shows an exemplary method of generating alloCTL when irradiatedlymphoblasts isolated from a brain tumor patient are mixed withperipheral blood mononuclear cells (PBMC) isolated from a healthy donorin a one-way mixed lymphocyte reaction (MLR).

FIG. 6 shows the percentage lysis achieved in one experiment fromChromium-51 (51 Cr)-release 4 hr assays at 3 effector to target (E:T)ratios (black, 20:1; red, 10:1; blue, 5:1).

FIG. 7 shows that, in some embodiments, specificity of alloCTL forrelevant glioma target can be demonstrated in 51 Cr-release cytotoxicityassays.

FIG. 8 shows an exemplary detection of apoptotic and necrotic cells bythe 7-amino actinomycin D (7AAD) assay.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The following detailed description relates to representative embodimentsof the invention. It is to be understood, however, that the presentinvention may be embodied in various forms. Therefore, the specificdetails disclosed herein are not to be interpreted as limiting, butrather as a representative basis for teaching one skilled in the art howto employ the present invention in virtually any detailed system,structure, or manner.

Brain tumor cells, such as glioma cells, express human leukocyteantigens (HLA, major histocompatibility complex antigens or MHC),whereas HLA are generally not expressed on normal, mitotically quiescentneuroglia. Therefore, the HLA expressed by the glioma cells can act astherapeutically useful tumor directed antigens. The lack of expressionof HLA antigens on normal brain tissues may limit the immune reactiononly to tumor cells. Plua, the relative immune privilege of the braincan extend the useful life-span of therapeutic alloCTL.

Alloreactive cytotoxic T lymphocytes (alloCTL) are T cells activatedagainst allogeneic HLA. The immune responses to major alloantigen arestronger than those engendered to minor tumor associated antigens (TAA),and the CTL precursor frequencies generally are higher to majoralloantigens than to TAA.

In one aspect, the present invention teaches methods and treatments, bywhich alloCTL adoptively transferred into an organ of a patient caninduce destruction of tumor cells, such as tumor cells (for example,brain tumor cells).

For generating robust alloCTLs, a responder:stimulator pairing ispredicted by methods and compositions described herein. Anyresponder:stimulator pairing prediction can be performed (i.e., ahealthy donor providing precursor CTL:patient). In one embodiment, apatient's irradiated white blood cells displaying HLA are used asstimulator while the donor's white blood cells are used as responders.Any prediction method can be used on any type of data from the patientand/or donor. For example, a prediction method can incorporate use of analgorithm, statistics, modeling, a simulation in vitro or in silico orany combination thereof.

Patient and Donor Antigen Information

Patient cell antigen information (also referred to herein as “patientantigen information”) and donor cell antigen information (also referredto herein as “donor antigen information”) can be any suitable antigeninformation useful for determining immunologic pairing for thepreparation of cytotoxic T cells.

In certain embodiments, major histocompatibility complex (MHC)information, which also is referred to as human leukocyte antigen (HLA)information, is provided. HLAs are encoded by the HLA loci on humanchromosome 6p. HLA information includes, without limitation, HLA class Iinformation, HLA class II information, a combination of both, and anyother suitable antigen information. HLA class I molecules often presentpeptides about 9 amino acids in length, and HLA class II molecules oftenpresent peptides about 15-24 amino acids in length. HLA class Imolecules often present peptides from within the cell, and HLA class IImolecules often present peptides from a source outside the cell that isbrought into the cell for presentation. An HLA molecule can interactwith a CD8+ activated T cell that recognizes the peptide presented bythe HLA molecule, and the T cell can kill the cell bearing the HLAmolecule with which the T cell interacts.

There are different groups of HLA class I molecules that include,without limitation, HLA-A, HLA-B, HLA-C, HLA-DR, DP, DQ; HLA-E, HLA-F,HLA-G, and HLA-K groups. Each group of HLA class I molecules includesmultiple alleles (one paternal and one fraternal). For example,HLA-A*0101, *0102, *0103, . . . *0130 are assigned to the serotype A1.The “A*01” prefix signifies that the gene products (expressed proteins)of the alleles are primarily identified by the A1 serotype or mostsimilar to alleles recognized by the serotype. There are differentgroups of HLA class II molecules that include, without limitation,HLA-DM, HLA-DQ, HLA-DP, HLA-DO and HLA-DR groups. Each group of class IImolecules encodes alpha-beta heterodimer proteins, and includes multiplealleles. For example, the HLA-DR group of HLA class II moleculesincludes DRB1*0101, DRB1*0102, DRB1*0103 and other alleles. Formammalian patients and donors (e.g., humans), each patient and donorcell bears two alleles in each group. Thus, patient and donor cells eachhave two HLA-A alleles, two HLA-B alleles and so on.

Patient and donor antigen information sometimes are referred to hereinas “antigen units,” and each antigen unit sometimes is an allele.Antigen information is one or more alleles in certain embodiments, andin some embodiments is between about 2 to about 38 alleles. Antigeninformation sometimes includes one allele for each HLA group provided,or both alleles of each HLA group provided. In some embodiments, antigeninformation includes one or two alleles from HLA groups (e.g., about1-19 HLA groups; about 2-18 groups).

Methods for determining an HLA allele are known in the art. For example,an HLA allele can be determined by methods that include, but are notlimited to, molecular typing, haplotyping, gene sequencing, cellulartyping and serotyping.

In molecular typing methods, for example, an amplification reaction(e.g., polymerase chain reaction, or PCR), can be utilized with sequencespecific primers (SSPs), where the size of an amplification product,and/or a sequence in or of an amplification product, can be assessed todetermine an HLA type (e.g., HLA allele). The latter method sometimes isreferred to as SSP-PCR when PCR is utilized as the amplificationprocess. A molecular typing method, in some embodiments, can involveidentification of a sequence in or of a product of an amplificationreaction (e.g., sequence base typing (SBT)). In SBT an amplificationproduct sometimes is immobilized and contacted with sequence specificprimers to determine a sequence of the product. Molecular typing alsocan be accomplished in some embodiments by a restriction fragment lengthpolymorphism (RFLP) method in which one or more amplification productsare digested with one or more enzymes, and the resulting fragments areanalyzed. In molecular typing methods that utilize an amplificationreaction, nested amplification reactions can be utilized in someembodiments. Haplotyping often involves determining multiple HLAs on onenucleic acid strand of a subject.

Gene sequencing methods generally involve sequencing all or a part of anHLA from a patient or donor using known sequencing methodology (e.g.,SBT-PCR). Serotyping often involves reacting cells from a patient ordonor with blood, antiserum and/or an antibody and determining which HLAantigens are present in the cell. In serotyping procedures, across-reacting HLA antigen can be recognized by monospecific antibodies(e.g., monoclonal or polyclonal) in certain embodiments. A cellulartyping method, such as a mixed lymphocyte culture (MLC or MLR) method,can be used to determine presence of an HLA allele by selectiveactivation of a particular T cell type. In some embodiments, a moleculartyping method (e.g., SSP-PCR, SBT and/or RFLP methods) is utilized togenerate antigen information for a donor and/or patient, and in certainembodiments, antigen information from a donor and/or a patient isobtained, or is complemented, with a cellular typing and/or cellulartyping method.

In some embodiments, antigen information is from a donor who isunrelated by family relationship to the patient. The donor may berelated by family relationship to the patient in certain embodiments,and may be, for example, a sibling, parent, grandparent, uncle, aunt,child, grandchild, niece or nephew of the patient. In some embodiments,the donor is not a sibling of the patient.

Stimulator and Responder Information

Stimulator information is related to patient cell antigen informationand responder information is related to donor cell antigen information.Stimulator information and responder information are related to antigeninformation, and often are derived or calculated from antigeninformation. Stimulator information and responder information includesamino acid differences, i.e., mismatch information derived fromcomparison of individual HLA alleles.

Stimulator information and responder information sometimes are T cellreceptor interaction information, which can be a peptide subsequencethat interacts, or is calculated to interact, with a T cell, in certainembodiments. Such information in the latter embodiments sometimes isreferred to as “eplet” information. Stimulator and responder informationsometimes is referred to herein as “stimulator/responder units,” andeach unit can be an eplet in certain embodiments.

Epitopes are antigenic determinants that elicit an immune response. Someepitopes are hidden (cryptotopes) that become immunologically availableafter fragmentation or denaturation of an antigen, for example. Aparatope involves a large group of surface residues that are involved inbinding to an antigen. There are two groups of protein epitopes, (1)continuous (or linear) epitopes involving a single continuous amino acidsequence and (2) discontinuous epitopes that comprise amino acidsseparated in the primary sequence but clustered together on themolecular surface by folding the native protein. Mapping studies ofantibody reactivity patterns with natural variants and mutated proteinantigens have generated information about the location of epitopes andhave also suggested that epitopes can generally be defined by smallnumbers of amino acid residues. A public epitope is an antigenic regionof amino acids that is antibody accessible. A private epitope is notaccessible by antibody, but may be accessable via HLA; cell receptors(TCR) interactions.

Eplets often are configurations of polymorphic amino acid surfaceresidues (triplets or patches) or small structural epitopes that play adominant role in determining recognition by a specific antibody. Theseresidues/triplets/patches often are within a 3 to 3.5 Angstrom radiusfrom each other. Stimulator and responder eplet information can providea more rigorous standard than serological HLA typing alone. Epletinformation along with other serological, protein and/or molecularinformation can also be used in combination.

Direct alloreactivity can be observed when T cells restricted to one HLAmolecule are exposed to antigen presenting cells bearing a peptidesequence from a different, but related HLA molecule. Many of thecontacts involved in T-cell receptor antigen recognition involve bindingof T-cell receptor elements to the HLA antigen-presenting face (forexample HLA alpha helices 1 and 2), and because of allelic structuraldifferences, the binding of the stimulator alloHLA to a T-cell receptoron a responder may be with greater affinity than to recognize self-HLA.Additionally, amino acid substitutions in the antigen (or peptide)binding groove of HLA can contribute to alloreactivity. In certainembodiments, methods described herein investigate HLA residues on theT-cell receptor “docking” face that are exposed and capable of takingpart in direct T-cell receptor binding, and HLA residues lining the HLAantigen-binding groove, that are capable of participating in antigenbinding. Since the enhanced affinity of the T-cell receptor for alloHLA+peptide may result from (a) changes in T-cell receptor/HLA interactions(i.e, HLA helices 1 and 2), (b) changes in T-cell receptor/peptideinteractions (i.e., peptide binding groove), or (c) a combination ofthese, an algorithm is used to look at these two classes of allelicchanges first separately, then in combination, seeking predictivecorrelations between structural differences and allostimulationpotential. In some embodiments, methods described herein are able todevelop “partial mismatch scoring” procedures for related HLA allelesthat will be predictive of alloresponses associated with the mismatch.Understanding of the structural and functional basis of T cellalloreactivity is useful in choosing responders that will providefunctionally robust alloCTLs based on HLA partial mismatch informationfrom stimulator and responders.

The assignment of antibody-accessible (B cell alloresponse occursbecause of three-dimensional or conformational differences), or T-cellreceptor accessible, positions is based on a detailed description of thecrystalline structure of various HLA class I and II molecules, likely onthe alpha helices and beta sheet of HLA. In certain embodiments,polymorphic triplets in the antibody-accessible positions of amino acidsequences are serologically defined as those that are recognized byalloantibody. Each triplet or patch is designated by its amino acidcomposition around a given position in the amino acid sequence.

Certain triplets/patches exhibit high immunogenicity whereas others haveintermediate or low immunogenicity. The total number of mismatches onthe face of the HLA and/or the type of aa differences is important fordetermining immunogenicity.

Comparison of stimulator and responder information can be based onlinear sequences of amino acids or patches of residues in linear anddiscontinuous sequences as motifs for potentially immunogenic epitopes.An HLA mismatch between the responder and stimulator is assessed bydetermining the number of amino acids not shared between the responderand stimulator's HLA antigens, in some embodiments.

The HLA molecules can be categorized into groups according to thepotential of being recognized as non-self or self when the patient isexposed to an HLA mismatch. For example, one group can consist oftriplets/patches that are present in one or two HLA antigens. Anothergroup can consist of polymorphic triplets/patches that are sharedbetween three or more HLA antigens encoded by the same class I or classII locus, for example. Another group can consist of triplets/patchesthat are polymorphic for one class I locus but monomorphic for anotherclass I locus, for example. Such triplets/patches may not representimmunogenic epitopes because they are always present on the patient'sown HLA antigens. Another group can consist of triplets/patches that arepolymorphic for one, two or all three HLA, A, B, and C loci for class Iantigens, for example. The translation of HLA with and expression bycells can also vary and influence immune response.

Stimulator and responder information also can be gained from in vitroexperimentation. Some molecules are produced at higher levels or lowerlevels in patients with rejected transplants or grafts. Some moleculesare produced at higher levels or lower levels in patients with stabletransplants or grafts. Assaying for these types of molecules in an invitro experiment can also generate stimulator and responder information.For example, a higher number of donor-specific interferon (IFN)-γproducing cells (proinflammatory, T-helper 1 cytokine) can be found inpatients with rejected transplants or grafts. Also a higher number ofinterleukin (IL)-10 producing cells (anti-inflammatory, T-helper 2cytokine) are found in patients with stable transplants or grafts. Byway of example, use of the number of IFN-γ producing cells and/orinterleukin IL-10 and/or a ratio of both can be used to identifyappropriate stimulators and/or responders.

Selection of a mismatch can depend on the antibody specificityrepertoire of the sensitized patient. HLA mismatch acceptability mayalso be assessed with information about the immunogencity of certainpolymorphisms. For example, highly sensitized patients can produce alimited repertoire of alloantibodies specific for the more common HLAepitopes. Although most highly sensitized patients have been exposed tomany mismatched alloantigens, their antibody reactivity patterns revealspecificity to a relatively small number of immunogenictriplets/patches, whereas other triplets/patches do not induce anantibody response and, therefore, must be non-immunogenic for thepatient. The generation and application of information aboutimmunogenicity of mismatched HLA can be used as stimulator/responderinformation.

Other stimulator/responder immunogenicity information which can beconsidered is the structural basis of antibody-antigen interactions suchas contact areas and binding energy. The binding energy of anantigen-antibody complex is primarily mediated by a small subset ofcontact residues in the epitope-paratope interface. Substitutions ofsuch “energetic” residues as seen in naturally occurring antigenicvariants or induced by site-directed or alanine scanning mutagenesislead often to dramatic decreases in the binding of antigen to antibody.Mapping studies have located energetic residues in “hot spots” ofepitopes and paratopes, i.e. regions made up of small numbers ofresidues that contribute most of the binding energy. Energetic residuesoften are located in the center of the epitope-paratope interface.

Other stimulator/responder immunogenicity information can include,without limitation, conventional serological cross-reactive group (CREG)mismatching (mm) (e.g., HLA-A, HLA-B, and any other suitable allele),any applicable/related HLA mismatching, HistoCheck, HLA-DR or DQmismatching, pretransplantation percent-reactive antibody (PRA),recipient and donor race and donor age, and cold ischemia time. Suchinformation can be used instead of eplet information or supplement epletinformation, in some embodiments.

The acronym HLA CREG refers to serological cross-reactive group to anyapplicable HLA molecule and describes how a monospecific HLA antiseracan react with two or more HLA antigens. The serologic cross-reactivityis assigned to determinants (public epitopes) that are differentiallyshared among HLA class I (or class II) gene products. For example, HLA-Aand HLA-B gene products can be grouped into eight or more families ofCREG based upon serologic cross-reactivity patterns, associativeanalyses, or shared amino acid sequence polymorphisms. Potentialresponders and stimulator pairs may be matched for public epitopes eventhough they are mismatched for the private epitopes that confer uniquedifferences between class I HLA molecules. Thus, there are levels ofimmunologic matching of HLA gene products ranging from the allele level,in which all public and private epitopes are matched, to the CREG level,in which public epitopes are matched but private epitopes aremismatched, in some embodiments.

The HistoCheck webtool similarly is a way of visualizing andunderstanding the structural differences among related majorhistocompatibility complex molecules. Because exact HLA matching oftenis not possible for organ transplant pairings, HistoCheck allows for theidentification of which alleles present the same structures (HLA—peptidecomplexes) to certain T-cell receptors despite having different aminoacid sequences. HistoCheck is a tool that provides a summary of aminoacid mismatches, positions, and functions as well as 3-dimensionalvisualizations.

The HistoCheck tool applies a distance index referred to as a Rislerindex. Similarity between single pairs of exchanged amino acids ismeasured by this distance matrix, as proposed by Risler J L, Delorme MO, Delacroix H, Henaut A, Amino acid substitutions in structurallyrelated proteins: A pattern recognition approach. Determination of a newand efficient scoring matrix, J Mol Biol 1988; 204: 1019-1029. A basicidea behind a Risler index score is that two distinct amino acids areless dissimilar the more often they are substituted for each other infunctionally related proteins. Accordingly, the fewer a pair of aminoacid substitutes each other, thus representing functional dissimilarity,the higher the score yielded, with the maximum value of 100.

Partial Mismatch and Methods for Identification

The presence or absence of a partial mismatch (e.g., one or moremismatches) between (i) patient antigen information and/or stimulatorinformation, and (ii) donor antigen information and/or responderinformation, often is identified in methods described herein. A partialmismatch is not a full match and often is a lower degree of matchingthan for an organ donor-patient pairing. A partial mismatch is a greaterdegree of matching than a total mismatch.

In embodiments where antigen units are compared, a partial mismatchsometimes is 1, 2, 3, 4, 5 or 6 patient/donor antigen units mismatchedshort of a full match in some embodiments, and in certain embodiments, apartial mismatch sometimes is 1, 2, 3, 4, 5 or 6 patient/donor antigenunits matched short of a full mismatch. In embodiments wherestimulator/responder units are compared, a partial mismatch sometimes is1, 2, 3, 4, 5 or 6 stimulator/responder units mismatched short of a fullmatch in some embodiments, and in certain embodiments, a partialmismatch sometimes is 1, 2, 3, 4, 5 or 6 stimulator/responder unitsmatched short of a full mismatch.

In certain embodiments, a partial mismatch is one or more amino aciddifferences between corresponding HLA molecules of a donor and patient(without limitation, 1-10 amino acids that differ between an HLA alleleof a donor and patient). A partial mismatch may be amino aciddifferences between one or more corresponding HLA types of a donor andpatient (e.g., one or more of HLA-A, HLA-B, HLA-C, HLA-DQ, HLA-DR,HLA-DP), where a corresponding HLA type is HLA-A allele of donor toHLA-A allele of patient, for example.

Identifying the presence or absence of a partial mismatch may beconducted by hand or by an algorithm, or a combination of the foregoing.In certain embodiments, provided are algorithms designed to identifypartial mismatches between stimulators and responders. Algorithms canmake use of the following information, in some embodiments: (1) each HLAantigen representing a distinct string of polymorphic triplets orpatches of residues in linear and discontinuous sequences as potentialimmunogens that can induce specific alloantibodies, and (2) sensitizedpatients do not have alloantibodies against triplets/patches present ontheir own HLA molecules. The algorithm can assess stimulator andresponder compatibility through intralocus and interlocus comparisonsand determine which amino acid mismatched HLA between donor and patient.This analysis can consider each responder (donor) HLA antigen mismatchtoward one or more particular HLAs in some embodiments, and in certainembodiments, a mismatch towards an entire class I (e.g., HLA-A, HLA-B,HLA-C) and/or class II (e.g., HLA-DR, HLA-DP, HLA-DQ) phenotype of thestimulator (patient), or subset thereof, in some embodiments. It shouldbe noted that, in general, gliomas express class I but little class IIantigen, so it may be inferred that the efficacy seen in patients in thesmall pilot study indicates class I CTL involvement.

The term “intralocus” as used herein refers to triplet/patch sharingbetween different HLA antigens encoded by the same locus, for examplecomparing loci on HLA-A from stimulator and responder. The term“interlocus” as used herein refers to triplet/patch sharing between HLAantigens encoded by different loci, for example comparison can be from aHLA-A locus from the stimulator and a HLA-B locus from the responder,for example. The latter includes triplets/patches that are polymorphicat one locus but monomorphic at another locus.

An algorithm can factor the structural basis of an HLA antigen partialmismatch utilizing intralocus and interlocus comparison of strings ofamino acid triplets/patches on antibody-accessible sties of HLA class Iand/or II molecules. The triplets/patches are elements of epitopes thatcan induce the formation of specific antibodies. This algorithm isdeveloped from stereochemical modeling of crystallized complexes ofantibodies with different protein antigens and published data about thecontributions of critical amino acid residues to antigen-antibodybinding energy. Three-dimensional structures of differentantigen-antibody complexes have revealed that up to six hypervariableloops (or complementarity determining regions) of the antibody bindingsite make contact with a protein antigen. Antigenic proteins havestructural epitopes consisting of 15-22 residues that constitute thebinding face with antibody. The surface of a structural epitope variesbetween 700 and 850 square Angstroms and is about the same as thesurface around the bound peptide-binding groove of an HLA molecule. Moststructural epitopes have a patch of about 2-5 so-called highly energeticresidues (sometimes referred to as ‘hot spots’) that dominate thestrength and specificity of binding with antibodies. The residues ofsuch functional epitopes are about 3 to 3.5 Angstroms apart from eachother and at least one of them is non-self. The remaining residues of astructural epitope contribute supplementary interactions that increasethe stability of the antigen-antibody complex.

In some embodiments, an algorithm applies the concept that each HLAantigen has multiple epitopes that can elicit specific alloantibodies.An algorithm also can address the total spectrum of antibody-accessibleamino acid sequence polymorphisms as critical components of potentiallyimmunogenic epitopes. An algorithm can consider a linear sequence ofthree amino acids as a minimal requirement for assessing HLAcompatibility at the molecular level. Partial mismatches are assessed bydetermining whether or not a triplet/patch in a given position of amismatched HLA antigen is also found in the same position in any of thepatient's own HLA alleles (e.g., HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP,HLA-DQ molecules, or subset thereof, or individual allele thereof). Ashared triplet/patch in the same position on a mismatched HLA antigenoften cannot elicit a specific antibody response in the patient.

The selection or assignment of triplets for matching purposes does notimply that the structural basis of an epitope always involves exactlythree amino acids. Many eplets or epitopes have only one or twopolymorphic residues, and some epitopes can be defined by four or fivepolymorphic residues in adjacent or distant positions.

In some embodiments, an algorithm can identify mismatched HLA antigensthat are fully compatible at the triplet/patch level. Many antigenscross react with the HLA antigens of the patient. In certainembodiments, a program can identify other cross-reacting antigens thatare incompatible at the triplet/patch level and can be used for partialmismatching.

The assignment of triplets/patches to HLA antigens may lack precision ifthe HLA typing information is based solely on serologic methods. Othermethods may reinforce HLA-typing information and be used with serologicmethods. For example, DNA-based typing can permit the definition of HLAsubtypes and, therefore, more accurate assignments of polymorphictriplets or patches. Many molecular subtypes of serologically definedHLA antigens have different triplets/patches in antibody-accessiblepositions. In such cases some serologically matched HLA antigens mayhave incompatible triplets/patches recognized by the patient'santibodies. In certain embodiments, stimulator information and/orresponder information can include serologic determinations of HLAantigen information, nucleic acid determinations of antigen information,or combinations of the forgoing.

An algorithm can be of any suitable type, including, without limitation,search algorithms, sorting algorithms, merge algorithms, numericalalgorithms, graph algorithms, string algorithms, modeling algorithms,computational genometric algorithms, combinatorial algorithms, machinelearning, cryptography, data compression algorithms and parsingtechniques and the like. An algorithm can comprise one or morealgorithms working in combination. An algorithm can be of any suitablecomplexity class and/or parameterized complexity. An algorithm can beused for calculation or data processing, or used in a deterministic orprobabilistic/predictive approach to a method in some embodiments.

Data Processing

The term “outcome” as used herein refers to the presence or absence of apartial mismatch (e.g., one or more mismatches) between (i) patientantigen information and/or stimulator information, and/or (ii) donorantigen information and/or responder information. Presence or absence ofan outcome can be expressed in any suitable form, including, withoutlimitation, ratio, deviation in ratio, frequency, distribution,probability (e.g., odds ratio, p-value), likelihood, percentage, valueover a threshold, or risk factor, associated with the presence of aoutcome for a subject or sample. Presence or absence of an outcome maybe identified based on one or more calculated variables, including, butnot limited to, ratio, distribution, frequency, sensitivity,specificity, standard deviation, coefficient of variation (CV), athreshold, confidence level, score, probability and/or a combinationthereof.

In certain embodiments, one or more of ratio, sensitivity, specificityand/or confidence level are expressed as a percentage. In someembodiments, the percentage, independently for each variable, is greaterthan about 90% (up to greater than 99%, even greater that 99.99%).Coefficient of variation (CV) in some embodiments is expressed as apercentage, and sometimes the percentage is about 10% or less (even lessthan 0.01%). A probability (e.g., that a particular outcome determinedby an algorithm is not due to chance) in certain embodiments isexpressed as a p-value, and sometimes the p-value is about 0.05 or less(even 0.000001 or less).

For example, scoring or a score may refer to calculating the probabilitythat a particular outcome is actually present or absent in astimulator/responder unit or pair. The value of a score may be used todetermine for example the variation, difference, or ratio of amplifiednucleic detectable product that may correspond to the actual outcome.For example, calculating a positive score from detectable eplets canlead to an identification of an outcome, which is particularly relevantto analysis of single patient or donor.

In certain embodiments, an algorithm can assign a confidence value tothe true positives, true negatives, false positives and false negativescalculated. The assignment of a likelihood of the occurrence of anoutcome can also be based on a certain probability model.

In certain embodiments, simulated (or simulation) data can aid dataprocessing, for example, by training an algorithm or testing analgorithm. Simulated data may for instance involve hypothetical varioussampling of different groupings of eplets intralocus or interlocus andthe like. Simulated data may be based on what might be expected from areal population or may be skewed to test an algorithm and/or to assign acorrect classification based on a simulated data set. Simulated dataalso is referred to herein as “virtual” data. Mismatch correlationswithin a stimulator/responder unit or pair can be simulated as a tableor array of numbers (for example, as a list of reactive eplets foundintralocus and interlocus between stimulator and responders), as agraph, as labeled intensity on a protein model, or as a representationof any technique that measures HLA partial mismatch distribution.Simulations can be performed in most instances by a computer program.One possible step in using a simulated data set is to evaluate theconfidence of the identified results, i.e. how well the selectedpositives/negatives match the sample and whether there are additionalvariations. A common approach is to calculate the probability value(p-value) which estimates the probability of a random sample havingbetter score than the selected one. As p-value calculations can beprohibitive in certain circumstances, an empirical model may beassessed, in which it is assumed that at least one sample matches areference sample (with or without resolved variations). Alternatively,other distributions such as Poisson distribution can be used to describethe probability distribution.

Simulated data often is generated in an in silico process. As usedherein, the term “in silico” refers to research and experimentsperformed using a computer. In silico methods include, but are notlimited to, molecular modeling studies, karyotyping, geneticcalculations, biomolecular docking experiments, and virtualrepresentations of molecular structures and/or processes, such asmolecular interactions.

As used herein, a “data processing routine” refers to a process that canbe embodied in software that determines the biological significance ofacquired data (i.e., the ultimate results of an assay). For example, adata processing routine can determine the amount of each nucleotidesequence species based upon the data collected. A data processingroutine also may control an instrument and/or a data collection routinebased upon results determined. A data processing routine and a datacollection routine often are integrated and provide feedback to operatedata acquisition by the instrument, and hence provide assay-basedjudging methods provided herein.

As used herein, software refers to computer readable programinstructions that, when executed by a computer, perform computeroperations. Typically, software is provided on a program productcontaining program instructions recorded on a computer readable medium,including, but not limited to, magnetic media including floppy disks,hard disks, and magnetic tape; and optical media including CD-ROM discs,DVD discs, magneto-optical discs, and other such media on which theprogram instructions can be recorded.

Different methods of predicting abnormality or normality can producedifferent types of results. For any given prediction, there are fourpossible types of outcomes: true positive, true negative, falsepositive, or false negative. The term “true positive” as used hereinrefers to a subject correctly diagnosed as having an outcome. The term“false positive” as used herein refers to a subject wrongly identifiedas having an outcome. The term “true negative” as used herein refers toa subject correctly identified as not having an outcome. The term “falsenegative” as used herein refers to a subject wrongly identified as nothaving an outcome. Two measures of performance for any given method canbe calculated based on the ratios of these occurrences: (i) asensitivity value, the fraction of predicted positives that arecorrectly identified as being positives (e.g., the fraction of matchedsets correctly identified by level comparison detection/determination asindicative of an outcome, relative to all matched sets identified assuch, correctly or incorrectly), thereby reflecting the accuracy of theresults in detecting the outcome; and (ii) a specificity value, thefraction of predicted negatives correctly identified as being negative(the fraction of matched sets correctly identified by level comparisondetection/determination as indicative of mismatching normality, relativeto all matched sets identified as such, correctly or incorrectly),thereby reflecting accuracy of the results in detecting the outcome.

The term “sensitivity” as used herein refers to the number of truepositives divided by the number of true positives plus the number offalse negatives, where sensitivity (sens) may be within the range of0≦sens≦1. Ideally, method embodiments herein have the number of falsenegatives equaling zero or close to equaling zero, so that no subject iswrongly identified as not having at least one outcome when they indeedhave at least one outcome. Conversely, an assessment often is made ofthe ability of a prediction algorithm to classify negatives correctly, acomplementary measurement to sensitivity. The term “specificity” as usedherein refers to the number of true negatives divided by the number oftrue negatives plus the number of false positives, where sensitivity(spec) may be within the range of 0≦spec≦1. Ideally, methods embodimentsherein have the number of false positives equaling zero or close toequaling zero, so that no subject wrongly identified as having at leastone outcome when they do not have the outcome being assessed. Hence, amethod that has sensitivity and specificity equaling one, or 100%,sometimes is selected.

One or more prediction algorithms may be used to determine significanceor give meaning to the detection data collected under variableconditions that may be weighed independently of or dependently on eachother. The term “variable” as used herein refers to a factor, quantity,or function of an algorithm that has a value or set of values. Forexample, a variable may be the age of the donor, age of the patient, sexof the patient, sex of the donor, ethnicity of the donor, ethnicity ofthe patient, number of eplets assessed, intralocus eplets assessed,interlocus eplets assessed and the like. The term “independent” as usedherein refers to not being influenced or not being controlled byanother. The term “dependent” as used herein refers to being influencedor controlled by another. For example, a particular eplet set beingassessed per each HLA types (A, B, or C) are variables that aredependent upon each other.

Any suitable type of method or prediction algorithm may be utilized togive significance to the data of the present invention within anacceptable sensitivity and/or specificity. For example, predictionalgorithms such as Mann-Whitney U Test, binomial test, log odds ratio,log-rank test, Chi-squared test, z-test, t-test, ANOVA (analysis ofvariance), regression analysis, neural nets, fuzzy logic, Hidden MarkovModels, multiple model state estimation, and the like may be used. Oneor more methods or prediction algorithms may be determined to givesignificance to the data having different independent and/or dependentvariables of the present invention. And one or more methods orprediction algorithms may be determined not to give significance to thedata having different independent and/or dependent variables of thepresent invention. One may design or change parameters of the differentvariables of methods described herein based on results of one or moreprediction algorithms (e.g., number of sets analyzed, types of eplets ineach set). For example, applying the Chi-square test to detection datamay suggest that specific HLA types are correlated to a higherlikelihood of having a particular brain tumor with a specific outcome,hence the variable of HLA types may be weighed differently versus beingweighed the same as other variables.

In certain embodiments, several algorithms may be chosen to be tested.These algorithms then can be trained with raw data. For each new rawdata sample, the trained algorithms will assign a classification to thatsample (i.e. partial mismatch). Based on the classifications of the newraw data samples, the trained algorithms' performance may be assessedbased on sensitivity and specificity. Finally, an algorithm with thehighest sensitivity and/or specificity or combination thereof may beidentified.

A sample is one or more cells from a donor or patient in someembodiments. Presence or absence of an outcome may be determined for allsamples tested, and in some embodiments, presence or absence of anoutcome is determined in a subset of the samples (e.g., samples fromGrade III tumor patients). In certain embodiments, an outcome isdetermined for about 60 to 99%, or even greater than 99%, of samplesanalyzed in a set. A set of samples can include any suitable number ofsamples, even more than 1000 samples. The set may be considered withrespect to samples tested in a particular period of time, and/or at aparticular location. The set may be partly defined by other criteria,for example, age and/or ethnicity. The set may be comprised of a samplewhich is subdivided into subsamples or replicates all or some of whichmay be tested. The set may comprise a sample from the same subjectcollected at two different times. In certain embodiments, an outcome isdetermined about 60% or more of the time for a given sample analyzed.

In certain embodiments, analyzing a higher number of characteristics(e.g., HLA antigens and/or DNA) that discriminate alleles can increasethe percentage of outcomes determined for the samples (e.g.,discriminated in a multiplex analysis). In some embodiments, one or moretissue or fluid samples (e.g., one or more blood samples) are providedby a subject (e.g., Grade III tumor patient). In certain embodiments,one or more RNA or DNA samples, or two or more replicate RNA or DNAsamples, are isolated from a single tissue or fluid sample, and analyzedby methods described herein.

As noted above, algorithms, software, processors and/or machines, forexample, can be utilized to (i) process detection data pertaining topartial mismatches, and/or (ii) identify the presence or absence of aoutcome. In certain embodiments, provided are methods for identifyingthe presence or absence of an outcome that comprise: (a) providing asystem, wherein the system comprises distinct software modules, andwherein the distinct software modules comprise a input module, a logicprocessing module, and a data display organization module; (b) detectinginput information indicating the presence or absence of a partialmismatch; (c) receiving, by the logic processing module, the inputinformation; (d) calling the presence or absence of an outcome by thelogic processing module; and (e) organizing, by the data displayorganization model in response to being called by the logic processingmodule, a data display indicating the presence or absence of theoutcome.

Provided also are methods for identifying the presence or absence of anoutcome, which comprise providing input information indicating thepresence or absence of a partial mismatch; providing a system, whereinthe system comprises distinct software modules, and wherein the distinctsoftware modules comprise an input detection module, a logic processingmodule, and a data display organization module; receiving, by the logicprocessing module, the input information; calling the presence orabsence of an outcome by the logic processing module; and, organizing,by the data display organization model in response to being called bythe logic processing module, a data display indicating the presence orabsence of the outcome.

Provided also are methods for identifying the presence or absence of anoutcome, which comprise providing a system, wherein the system comprisesdistinct software modules, and wherein the distinct software modulescomprise an input detection module, a logic processing module, and adata display organization module; receiving, by the logic processingmodule, input information indicating the presence or absence of apartial mismatch; calling the presence or absence of an outcome by thelogic processing module; and, organizing, by the data displayorganization model in response to being called by the logic processingmodule, a data display indicating the presence or absence of theoutcome.

By “providing input information” is meant any manner of providing theinformation, including, for example, computer communication means from alocal, or remote site, human data entry, or any other method oftransmitting input information. The signal information may be generatedin one location and provided to another location.

By “obtaining” or “receiving” input information is meant receiving thesignal information by computer communication means from a local, orremote site, human data entry, or any other method of receiving signalinformation. The input information may be generated in the same locationat which it is received, or it may be generated in a different locationand transmitted to the receiving location.

By “indicating” or “representing” the amount is meant that the inputinformation is related to, or correlates with, for example, the percentmismatch or presence or absence of partial mismatch. The information maybe, for example, the calculated data associated with the presence orabsence of partial mismatch as obtained, for example, after convertingraw data obtained by HLA typing.

Also provided are computer program products, such as, for example, acomputer program product comprising a computer usable medium having acomputer readable program code embodied therein, the computer readableprogram code adapted to be executed to implement a method foridentifying the presence or absence of an outcome, which comprises (a)providing a system, wherein the system comprises distinct softwaremodules, and wherein the distinct software modules comprise a signaldetection module, a logic processing module, and a data displayorganization module; (b) detecting input information indicating thepresence or absence of a partial mismatch; (c) receiving, by the logicprocessing module, the input information; (d) calling the presence orabsence of an outcome by the logic processing module; and, organizing,by the data display organization model in response to being called bythe logic processing module, a data display indicating the presence orabsence of the outcome.

Also provided are computer program product, such as, for example,computer program products comprising a computer usable medium having acomputer readable program code embodied therein, the computer readableprogram code adapted to be executed to implement a method foridentifying the presence or absence of an outcome, which comprisesproviding a system, wherein the system comprises distinct softwaremodules, and wherein the distinct software modules comprise a signaldetection module, a logic processing module, and a data displayorganization module; receiving signal information indicating thepresence or absence of a partial mismatch; calling the presence orabsence of an outcome by the logic processing module; and, organizing,by the data display organization model in response to being called bythe logic processing module, a data display indicating the presence orabsence of the outcome.

Input information may be, for example, total number of mismatches,specific types of mismatches, or both must be factored into development,i.e., training and validation of the program along with in vitrofunctional and phenotypic data obtained from an algorithm, orstatistical likelihood given other parameters. The mismatch data may beraw data, such as, for example, a set of numbers, or, for example, arange of mismatch dependent upon HLA type. The input information may beconverted or transformed to any form of data that may be provided to, orreceived by, a computer system. The input information may also, forexample, be converted, or transformed to identification data orinformation representing an outcome. An outcome may be, for example, aspecific HLA type, a HLA type ratio, or a particular percentagemismatch, for example.

Also provided is a machine for identifying the presence or absence of anoutcome wherein the machine comprises a computer system having distinctsoftware modules, and wherein the distinct software modules comprise asignal detection module, a logic processing module, and a data displayorganization module, wherein the software modules are adapted to beexecuted to implement a method for identifying the presence or absenceof an outcome, which comprises (a) detecting input informationindicating the presence or absence of a partial mismatch; (b) receiving,by the logic processing module, the signal information; (c) calling thepresence or absence of an outcome by the logic processing module,wherein a percent partial mismatch different than a normal matching ormismatching ratio is indicative of a good stimulator/responder unit; and(d) organizing, by the data display organization model in response tobeing called by the logic processing module, a data display indicatingthe presence or absence of the outcome. The machine may further comprisea memory module for storing signal information or data indicating thepresence or absence of a partial mismatch. Also provided are methods foridentifying the presence or absence of an outcome, wherein the methodscomprise the use of a machine for identifying the presence or absence ofan outcome.

Also provided are methods for identifying the presence or absence of anoutcome that comprises: (a) detecting input information, wherein theinput information indicates presence or absence of a partial mismatch;(b) transforming the input information into identification data, whereinthe identification data represents the presence or absence of theoutcome, whereby the presence or absence of the outcome is identifiedbased on the signal information; and (c) displaying the identificationdata.

Also provided are methods for identifying the presence or absence of anoutcome that comprises: (a) providing signal information indicating thepresence or absence of a partial mismatch; (b) transforming the signalinformation representing into identification data, wherein theidentification data represents the presence or absence of the outcome,whereby the presence or absence of the outcome is identified based onthe signal information; and (c) displaying the identification data.

Also provided are methods for identifying the presence or absence of anoutcome that comprises: (a) receiving signal information indicating thepresence or absence of a partial mismatch; (b) transforming the signalinformation into identification data, wherein the identification datarepresents the presence or absence of the outcome, whereby the presenceor absence of the outcome is identified based on the signal information;and (c) displaying the identification data.

For purposes of these, and similar embodiments, the term “inputinformation” indicates information readable by any electronic media,including, for example, computers that represent data derived using thepresent methods. For example, “input information” can represent theamount of a partial mismatch or percentage. Input information, such asin these examples, that represents physical substances may betransformed into identification data, such as a visual display, thatrepresents other physical substances, such as, for example, a HLAdisorder, or a HLA type. Identification data may be displayed in anyappropriate manner, including, but not limited to, in a computer visualdisplay, by encoding the identification data into computer readablemedia that may, for example, be transferred to another electronic device(e.g., electronic record), or by creating a hard copy of the display,such as a print out or physical record of information. The informationmay also be displayed by auditory signal or any other means ofinformation communication. In some embodiments, the input informationmay be detection data obtained using methods to detect a partialmismatch.

Once the input information is detected, it may be forwarded to thelogic-processing module. The logic-processing module may “call” or“identify” the presence or absence of an outcome.

Provided also are methods for transmitting genetic information to asubject, which comprise identifying the presence or absence of anoutcome wherein the presence or absence of the outcome has beendetermined from determining the presence or absence of a partialmismatch from a sample from the subject; and transmitting the presenceor absence of the outcome to the subject. A method may includetransmitting HLA type information of a brain tumor subject and donor,and the outcome may be presence or absence of a partial mismatch betweenthe two, in certain embodiments.

The term “identifying the presence or absence of an outcome” or “anincreased risk of an outcome,” as used herein refers to any method forobtaining such information, including, without limitation, obtaining theinformation from a laboratory file. A laboratory file can be generatedby a laboratory that carried out an assay to determine the presence orabsence of an outcome. The laboratory may be in the same location ordifferent location (e.g., in another country) as the personnelidentifying the presence or absence of the outcome from the laboratoryfile. For example, the laboratory file can be generated in one locationand transmitted to another location in which the information thereinwill be transmitted to the subject. The laboratory file may be intangible form or electronic form (e.g., computer readable form), incertain embodiments.

The term “transmitting the presence or absence of the outcome to thesubject” or any other information transmitted as used herein refers tocommunicating the information to the subject, or family member, guardianor designee thereof, in a suitable medium, including, withoutlimitation, in verbal, document, or file form.

Also provided are methods for providing to a subject a medicalprescription based on genetic information, which comprise identifyingthe presence or absence of an outcome, wherein the presence or absenceof the outcome has been determined from the presence or absence of apartial mismatch from a stimulator/responder unit; and providing amedical prescription based on the presence or absence of the outcome tothe patient. The medical prescription is administration of alloreactivecytotoxic T cells prepared from responder/stimulator pair identified bya partial mismatch, in some embodiments.

Also provided are files, such as, for example, a file comprising thepresence or absence of outcome for a subject, wherein the presence orabsence of the outcome has been determined from the presence or absenceof a partial mismatch from a stimulator/responder unit. The file may be,for example, but not limited to, a computer readable file, a paper file,or a medical record file.

Computer program products include, for example, any electronic storagemedium that may be used to provide instructions to a computer, such as,for example, a removable storage device, CD-ROMS, a hard disk installedin hard disk drive, signals, magnetic tape, DVDs, optical disks, flashdrives, RAM or floppy disk, and the like.

Systems discussed herein may further comprise general components ofcomputer systems, such as, for example, network servers, laptop systems,desktop systems, handheld systems, personal digital assistants,computing kiosks, and the like. The computer system may comprise one ormore input means such as a keyboard, touch screen, mouse, voicerecognition or other means to allow the user to enter data into thesystem. The system may further comprise one or more output means such asa CRT or LCD display screen, speaker, FAX machine, impact printer,inkjet printer, black and white or color laser printer or other means ofproviding visual, auditory or hardcopy output of information.

Input and output devices may be connected to a central processing unitwhich may comprise among other components, a microprocessor forexecuting program instructions and memory for storing program code anddata. In some embodiments the methods may be implemented as a singleuser system located in a single geographical site. In other embodimentsmethods may be implemented as a multi-user system. In the case of amulti-user implementation, multiple central processing units may beconnected by means of a network. The network may be local, encompassinga single department in one portion of a building, an entire building,span multiple buildings, span a region, span an entire country or beworldwide. The network may be private, being owned and controlled by theprovider or it may be implemented as an Internet based service where theuser accesses a web page to enter and retrieve information.

The various software modules associated with the implementation of thepresent products and methods can be suitably loaded into the a computersystem as desired, or the software code can be stored on acomputer-readable medium such as a floppy disk, magnetic tape, or anoptical disk, or the like. In an online implementation, a server and website maintained by an organization can be configured to provide softwaredownloads to remote users. As used herein, “module,” includinggrammatical variations thereof, means, a self-contained functional unitwhich is used with a larger system. For example, a software module is apart of a program that performs a particular task. Thus, provided hereinis a machine comprising one or more software modules described herein,where the machine can be, but is not limited to, a computer (e.g.,server) having a storage device such as floppy disk, magnetic tape,optical disk, random access memory and/or hard disk drive, for example.

The present methods may be implemented using hardware, software or acombination thereof and may be implemented in a computer system or otherprocessing system. An example computer system may include one or moreprocessors. A processor can be connected to a communication bus. Thecomputer system may include a main memory, sometimes random accessmemory (RAM), and can also include a secondary memory. The secondarymemory can include, for example, a hard disk drive and/or a removablestorage drive, representing a floppy disk drive, a magnetic tape drive,an optical disk drive, memory card etc. The removable storage drivereads from and/or writes to a removable storage unit in a well-knownmanner. A removable storage unit includes, but is not limited to, afloppy disk, magnetic tape, optical disk, etc. which is read by andwritten to by, for example, a removable storage drive. As will beappreciated, the removable storage unit includes a computer usablestorage medium having stored therein computer software and/or data.

In certain embodiments, secondary memory may include other similar meansfor allowing computer programs or other instructions to be loaded into acomputer system. Such means can include, for example, a removablestorage unit and an interface device. Examples of such can include aprogram cartridge and cartridge interface (such as that found in videogame devices), a removable memory chip (such as an EPROM, or PROM) andassociated socket, and other removable storage units and interfaceswhich allow software and data to be transferred from the removablestorage unit to a computer system.

A computer system may also include a communications interface. Acommunications interface allows software and data to be transferredbetween the computer system and external devices. Examples ofcommunications interface can include a modem, a network interface (suchas an Ethernet card), a communications port, a PCMCIA slot and card,etc. Software and data transferred via communications interface are inthe form of signals, which can be electronic, electromagnetic, opticalor other signals capable of being received by communications interface.These signals are provided to communications interface via a channel.This channel carries signals and can be implemented using wire or cable,fiber optics, a phone line, a cellular phone link, an RF link and othercommunications channels. Thus, in one example, a communicationsinterface may be used to receive signal information to be detected bythe signal detection module.

In a related aspect, the signal information may be input by a variety ofmeans, including but not limited to, manual input devices or direct dataentry devices (DDEs). For example, manual devices may include,keyboards, concept keyboards, touch sensitive screens, light pens,mouse, tracker balls, joysticks, graphic tablets, scanners, digitalcameras, video digitizers and voice recognition devices. DDEs mayinclude, for example, bar code readers, magnetic strip codes, smartcards, magnetic ink character recognition, optical characterrecognition, optical mark recognition, and turnaround documents. In oneembodiment, an output from a gene or chip reader my serve as an inputsignal.

FIG. 2 shows a flowchart 200 generally outlining an embodiment of amethod described herein. In FIG. 2, collection of stimulator and/orresponder information (210) contributes to identification (220) of thepresence or absence of a partial mismatch for stimulator andresponder(s) pairs. Identification (220) can be performed by analgorithm, statistics, modeling, a simulation in vitro or in silico orany combination thereof. Identification (220) of the presence of apartial mismatch leads to combining cells of a selected stimulator andresponder(s). After combining stimulator and responder cells (230), invitro immunogenic data optionally may be collected and used to alter themethod for performing identification (220) (e.g., the method comprisesan algorithm) and/or to improve identification of partial mismatches,shown by arrow 270. After combining stimulator and responder cells(230), cytotoxic T lymphocytes are harvested (240) and administered tothe patient/stimulator (250). Optionally, the patient/stimulator'sresponse to the treatment is monitored (260) and in vivo data optionallyis collected (e.g., in vivo immunogenic data, anti-tumor response).Optionally, in vivo immunogenic data is used to alter the method forperforming identification (220) (e.g., the method comprises analgorithm) and/or to improve identification of partial mismatches, shownby arrow 280.

FIG. 3 shows a flowchart 300 generally outlining a method describedherein, where stimulator and responder information or data collected 310is used by any known data processing method 320, such as for example analgorithm, statistics, modeling, a simulation in vitro or in silico orany combination thereof. Data 320 is used to identify a partial mismatch330 of stimulator and/or responder information.

A non-limiting example of how a process in FIG. 3 can occur is with DNA,RNA, or protein structure information on HLA molecules from stimulatorand responders. After HLA typing each stimulator and responder, mismatchinformation between each HLA molecule can be assessed. Identification ofmismatched amino acids can be determined from HLA antigen information,such as, molecularly and/or serologically determined HLA-A, -B, -C, -DR,-DP, -DQ molecules (e.g., individual alleles or antigens, groups ofalleles or antigens of all alleles or antigens), for example. FurtherHLA analysis, such as location, surface expression, and/or amino acidcomposition, of each mismatched aa also can be generated. Identificationof highly immunogenic mismatches can increase activation of cytotoxic Tcells.

Once the 3D structural HLA protein information is processed into 2Damino acid composition, then any data processing 320 can occur toproduce identification of partial mismatches 330. For example, aminoacid composition of HLA molecules for stimulator and responders can beprocessed through a string matching algorithm where the amino acidpattern for a particular HLA molecule (e.g., per mismatch, group ofmismatches, HLA class, HLA molecule type, or the like) is used andsearched for other identical or similar occurrences or locations withinthe same molecule, different molecule and/or different sample. Thealgorithm can process data in any known way, for example by hand or byusing a computing environment as depicted in FIG. 4.

Any processing of data 320, such as using an algorithm, can be utilizedin a computing environment, such as FIG. 4, by use of a programminglanguage such as C, C++, Java, Perl, Python, Fortran and the like. Thealgorithm can be modified to include margin of errors, statisticanalysis, in vivo data 280 and in vitro data 270 as well as comparisonto other stimulator/responder information (for example in using a neuralnet or clustering algorithm). The algorithm can then assign matching,mismatching and/or partial mismatching of aa per HLA molecule forstimulator and responder(s) pairs.

FIG. 4 illustrates a non-limiting example of a computing environment 610in which various systems, methods, algorithms, and data structuresdescribed herein may be implemented. The computing environment 610 isonly one example of a suitable computing environment and is not intendedto suggest any limitation as to the scope of use or functionality of thesystems, methods, and data structures described herein. Neither shouldcomputing environment 610 be interpreted as having any dependency orrequirement relating to any one or combination of components illustratedin computing environment 610. A subset of systems, methods, and datastructures shown in FIG. 4 can be utilized in certain embodiments.

Systems, methods, and data structures described herein are operationalwith numerous other general purpose or special purpose computing systemenvironments or configurations. Examples of known computing systems,environments, and/or configurations that may be suitable include, butare not limited to, personal computers, server computers, thin clients,thick clients, hand-held or laptop devices, multiprocessor systems,microprocessor-based systems, set top boxes, programmable consumerelectronics, network PCs, minicomputers, mainframe computers,distributed computing environments that include any of the above systemsor devices, and the like.

The operating environment 610 of FIG. 4 includes a general purposecomputing device in the form of a computer 620, including a processingunit 621, a system memory 622, and a system bus 623 that operativelycouples various system components include the system memory to theprocessing unit 621. There may be only one or there may be more than oneprocessing unit 621, such that the processor of computer 620 comprises asingle central-processing unit (CPU), or a plurality of processingunits, commonly referred to as a parallel processing environment. Thecomputer 620 may be a conventional computer, a distributed computer, orany other type of computer.

The system bus 623 may be any of several types of bus structuresincluding a memory bus or memory controller, a peripheral bus, and alocal bus using any of a variety of bus architectures. The system memorymay also be referred to as simply the memory, and includes read onlymemory (ROM) 624 and random access memory (RAM) 625. A basicinput/output system (BIOS) 626, containing the basic routines that helpto transfer information between elements within the computer 620, suchas during start-up, is stored in ROM 624. The computer 620 may furtherinclude a hard disk drive interface 627 for reading from and writing toa hard disk, not shown, a magnetic disk drive 628 for reading from orwriting to a removable magnetic disk 629, and an optical disk drive 630for reading from or writing to a removable optical disk 631 such as a CDROM or other optical media.

The hard disk drive 627, magnetic disk drive 628, and optical disk drive630 are connected to the system bus 623 by a hard disk drive interface632, a magnetic disk drive interface 633, and an optical disk driveinterface 634, respectively. The drives and their associatedcomputer-readable media provide nonvolatile storage of computer-readableinstructions, data structures, program modules and other data for thecomputer 620. Any type of computer-readable media that can store datathat is accessible by a computer, such as magnetic cassettes, flashmemory cards, digital video disks, Bernoulli cartridges, random accessmemories (RAMs), read only memories (ROMs), and the like, may be used inthe operating environment.

A number of program modules may be stored on the hard disk, magneticdisk 629, optical disk 631, ROM 624, or RAM 625, including an operatingsystem 635, one or more application programs 636, other program modules637, and program data 638. A user may enter commands and informationinto the personal computer 620 through input devices such as a keyboard40 and pointing device 642. Other input devices (not shown) may includea microphone, joystick, game pad, satellite dish, scanner, or the like.These and other input devices are often connected to the processing unit621 through a serial port interface 646 that is coupled to the systembus, but may be connected by other interfaces, such as a parallel port,game port, or a universal serial bus (USB). A monitor 647 or other typeof display device is also connected to the system bus 623 via aninterface, such as a video adapter 648. In addition to the monitor,computers typically include other peripheral output devices (not shown),such as speakers and printers.

The computer 620 may operate in a networked environment using logicalconnections to one or more remote computers, such as remote computer649. These logical connections may be achieved by a communication devicecoupled to or a part of the computer 620, or in other manners. Theremote computer 649 may be another computer, a server, a router, anetwork PC, a client, a peer device or other common network node, andtypically includes many or all of the elements described above relativeto the computer 620, although only a memory storage device 650 has beenillustrated in FIG. 4. The logical connections depicted in FIG. 4include a local-area network (LAN) 651 and a wide-area network (WAN)652. Such networking environments are commonplace in office networks,enterprise-wide computer networks, intranets and the Internet, which allare types of networks.

When used in a LAN-networking environment, the computer 620 is connectedto the local network 651 through a network interface or adapter 653,which is one type of communications device. When used in aWAN-networking environment, the computer 620 often includes a modem 654,a type of communications device, or any other type of communicationsdevice for establishing communications over the wide area network 652.The modem 654, which may be internal or external, is connected to thesystem bus 623 via the serial port interface 646. In a networkedenvironment, program modules depicted relative to the personal computer620, or portions thereof, may be stored in the remote memory storagedevice. It is appreciated that the network connections shown arenon-limiting examples and other communications devices for establishinga communications link between computers may be used.

Stimulator Cell and Donor Cell Preparation

After identifying the presence of a partial mismatch for a donor/patientpair, cytotoxic T cells may be prepared by mixing cells of the donorwith inactivated cells of the patient for donor/patient pairs exhibitinga partial mismatch. Stimulator cells and responder cells are preparedbefore such an activation reaction is conducted.

Stimulator cells, which are derived from a patient, and responder cells,which are derived from a donor, independently can be from any suitablesource. A source of cells includes, without limitation, blood, bloodfraction (e.g., plasma, serum, buffy coat, red blood cell layer), bonemarrow, biological fluid (e.g., urine, blood, saliva, amniotic fluid,exudate from a region of infection or inflammation, mouth wash, cerebralspinal fluid, synovial fluid), or organ, tissue, cell, cell pellet, cellextract or biopsy (e.g., brain, neck, spine, throat, heart, lung,breast, kidney, liver, intestine, colon, pancreas, bladder, cervix,testes, skin and the like). The source can be directly from the patientor donor, sometimes is frozen, and at times is provided as a cellsuspension. A source of cells includes, without limitation, a human oran animal (e.g., canine, feline, ungulate (e.g., equine, bovine,caprine, ovine, porcine, buffalo, camel and the like), rodent (e.g.,murine, mouse, rat), avian, amphibian, reptile, fish).

Cells from a patient sometimes are from patient blood, and in certainembodiments are immune cells, such as simulator white blood cells orlymphocytes or dendritic cells from the blood. Cells from a donorsometimes are from donor blood, and in certain embodiments are whiteblood cells or lymphocytes from the blood. Stimulator donor blood and orbuffy coat sometimes is from a blood bank. Blood sometimes is peripheralblood, sometimes is a blood fraction (e.g., buffy coat), sometimes iszero to seven days old, and at times is frozen blood or frozen bloodfraction (e.g., blood cells are vitally cryopreserved).

A patient from whom stimulator cells are derived often is afflicted witha medical condition. A medical condition can be a cell proliferationcondition, an autoimmune condition and/or inflammation condition, insome embodiments (non-limiting examples are provided herein).

Donor cells or patient cells, or stimulator cells or responder cells,sometimes include a substantial amount of a particular type of cell. Theterm “substantial amount” as used in the foregoing sentence refers to25% or more of cells in a container (e.g., flask, tube, plate; about 30%or more). Particular cell types include, without limitation, white bloodcell, granulocyte, agranulocyte, monocyte, lymphocyte, B cell, T cell,CD4+ T cell, CD8+ T cell, natural killer cell, stem cell (e.g., CD34+cell), lymphoblast, antigen presenting cell, dendritic cell, macrophage,neutrophil, eosinophil, basophil. An antigen presenting cell sometimesis a professional antigen presenting cell, which can include, withoutlimitation, a dendritic cell, macrophage, B cell and activatedepithelial cell.

Donor cells and/or patient cells sometimes are subjected to a treatmentprocess before they are combined for activation of T cells intocytotoxic T cells. A treatment process can increase the relative amountof a particular cell type in a composition, or can generate a new celltype in a population. For example, a treatment process can be utilizedto differentiate patient cells into dendritic cells or activate patientcells into lymphoblasts, in certain embodiments. Certain treatments ofdonor cells into stimulator cells can improve the immunogenic action ofresponder cells when the stimulator cells are combined with theresponder cells.

In some embodiments, however, donor cells and/or patient cells are notsubjected to a treatment process prior to combining them with oneanother for production of cytotoxic T cells (e.g., white blood cellsfrom the donor are mixed with stimulator cells). In the latterembodiments, the donor cells and patient cells are responder cells andstimulator cells, respectively.

In certain treatment methods, white blood cells from a patient or donorare provided and certain cell types are separated. White blood cellssometimes are collected by isolating peripheral blood mononuclear cells(PBMC) by a suitable method (e.g., ficoll gradient centrifugation). Insome embodiments, monocytes are separated (e.g., for differentiationinto dendritic cells), and sometimes are separated from othernonadherent cells because they adhere to a solid support in a particularmedium (e.g., AIM-V medium) in certain embodiments. Lymphocytes areseparated (e.g., for activation of lymphoblasts) in some embodiments,and sometimes are separated by collecting cells that do not adhere to asolid support in a particular medium (e.g., commercially available AIM-Vmedium).

In some embodiments, a treatment method prepares dendritic cells (DCs).Dendritic cells can be prepared by any suitable method known in the art,and non-limiting examples of DC differentiation methods are describedherein (e.g., Examples section). In some embodiments, DCs are separatedfrom other cells in a population and then expanded. In such methods, DCsmay be contacted with one or more antibodies that bind to DC cellmarkers, and the DCs are separated by flow cytometry, in certainembodiments.

In some embodiments, DCs are differentiated from precursor cells. Insome DC differentiation methods, monocytes from PBMC are differentiatedinto immature DCs and then to mature DCs. Immature DCs sometimes aredifferentiated from monocytes by contacting the latter with one or moresuitable stimulants. Any suitable medium can be utilized fordifferentiation of dendritic cells (e.g., AIM-V medium). In certainembodiments, DCs are differentiated from stem cells. DCs derived from apatient and selected for combination with donor cells are of anysuitable maturation or activation state and can express Toll-likereceptors of various types. Cultures having mature DCs are selected forcombination with donor cells in certain embodiments.

Examples of stimulants include, without limitation, cytokines, whichinclude, for example, interleukins (e.g., IL-1-IL-18 and the like),interferons (e.g., IFN-α, IFN-β, IFN-γ and the like), cytokins (e.g.,TNF-α, TNF-β and the like), lymphokines, monokines and chemokines;growth factors (e.g., transforming growth factors (e.g., TGF-alpha,TGF-beta and the like)); colony-stimulating factors (e.g., granulocytemacrophage colony-simulating factor (GM-CSF), granulocytecolony-simulating factor (G-CSF) etc.); and the like. In someembodiments, monocytes are contacted with one or more interleukins(e.g., IL-4), and/or one or more colony-stimulating factors (e.g.,GM-CSF). In certain embodiments, monocytes and/or immature DCs arecontacted with one or more interleukins (e.g., IL-6, IL-1beta) and/orone or more tumor necrosis factors (e.g., TNF alpha). A suitable amountof stimulant is selected as known in the art, and the amount of astimulant may range from about 5 units to about 5000 units (e.g.,International Units). In some embodiments, about 0.2 ng/ml to about 1000ng/ml of a stimulant is utilized. A stimulant can be native polypeptidepurified from a cell and often is recombinant polypeptide. A stimulantoften is a human polypeptide, and often is produced by recombinantmethods (e.g., recombinant human IL-2 (rhIL-2)).

A DC can be differentiated from a stem cell in some embodiments. Incertain non-limiting DC differentiation methods, a hematopoietic stemcell (e.g., a human CD34+ stem cell) can be differentiated into adendritic cell. Stem cells can be isolated by methods known in the art.For example, bone marrow aspirations from iliac crests can be performede.g., under general anesthesia in the operating room. The bone marrowaspiration sometimes is approximately 1,000 ml in quantity and often iscollected from the posterior iliac bones and crests. If the total numberof cells collected is less than about 2×10⁸/kg, a second aspiration isoptionally performed (e.g., using the sternum and/or anterior iliaccrests in addition to posterior crests). During the operation, two unitsof irradiated packed red cells can be administered to replace the volumeof marrow taken by the aspiration. Human hematopoietic progenitor cellsand stem cells can be characterized by the presence of a CD34 surfacemembrane antigen. This antigen often is used for purification. After thebone marrow is harvested, the mononuclear cells can be separated fromother components by ficol gradient centrifugation. This centrifugationcan be performed by a semi-automated method using a cell separator(e.g., a Baxter Fenwal CS3000+ or Terumo machine). The light densitycells, composed mostly of mononuclear cells, are collected and the cellsare incubated in plastic flasks at 37° C. for 1.5 hours. The adherentcells (e.g., monocytes, macrophages and B-Cells) often are discarded.The non-adherent cells can be collected can be incubated with amonoclonal anti-CD34 antibody (e.g., the murine antibody 9C5) at 4° C.for 30 minutes with gentle rotation. The final concentration for theanti-CD34 antibody often is 10 micrograms/ml. After two washes,paramagnetic microspheres (Dyna Beads, supplied by Baxter ImmunotherapyGroup, Santa Ana, Calif.) coated with sheep antimouse IgG (Fc) antibodycan be added to the cell suspension at a ratio of 2 cells/bead. After afurther incubation period of 30 minutes at 4° C., the rosetted cellswith magnetic beads are collected with a magnet. Chymopapain (suppliedby Baxter Immunotherapy Group, Santa Ana, Calif.) at a finalconcentration of 200 U/ml can be added to release the beads from theCD34+ cells. Alternatively, an affinity column isolation procedure canbe used which binds to CD34, or to antibodies bound to CD34.

Stem cells can be differentiated in vitro using appropriate cytokines(e.g., GM-CSF). The concentration of GM-CSF in culture can be about 0.2ng/ml or more, sometimes about 1 ng/ml or more, and at times betweenabout 20 ng/ml and about 200 ng/ml (e.g., about 100 ng/ml), in certainembodiments. In some embodiments, TNF-alpha also is added to facilitatedifferentiation, sometimes in about the same concentration range as forGM-CSF. Optionally, a proliferation ligand (e.g., stem cell factor(SCF), Flt 3 ligand) is added in similar concentration ranges todifferentiate human DCs, and in some embodiments, IL-4 is added insimilar ranges to promote DC differentiation. In certain embodiments, aDC or DC precursor cell is transduced with a nucleic acid. The nucleicacid may encode an interleukin and/or a colony-stimulating factor (e.g.,IL-4 and/or GM-CSF; U.S. Pat. No. 7,378,277, Hwu et al.). Atransduction-facilitating agent (e.g., lipofectamine) can be introducedto facilitate nucleic acid transfer to cultured cells. Optimizedconcentrations of stimulants described in this paragraph can be assessedby titrating stimulant and observing effects (e.g., U.S. Pat. No.7,378,277, supra).

In certain non-limiting DC differentiation methods, peripheral bloodmononuclear cells (PBMC) from healthy donors can be can be isolated bydensity centrifugation of heparinized blood on Lymphoprep (Nycomed,Oslo, Norway). PBMC can be washed with PBS, resuspended in CellGenix DCmedium (Freiburg, Germany) and allowed to adhere in culture plates for 2h at 37° C. and 5% CO₂. Nonadherent cells can be removed by extensivewashings, and adherent monocytes can be cultured for 5 days in thepresence of 500 U/ml hIL-4 and 800 U/ml hGM-CSF (R&D Systems,Minneapolis, Minn.). As assessed by morphology and FACS analysis,resulting immature DCs (imDCs) often are MHC-class I, IIhi, and oftenexpress CD401o, CD801o, CD831o, and/or CD861o. Immature DCs often areCD14 neg and contain less than 3% of contaminating CD3+ T, CD19+ B, andCD16+ NK cells. DCs can be stimulated with MPL, FSL-1, Pam₃CSK₄(InvivoGen, San Diego, Calif.), LPS (Sigma-Aldrich, St. Loucan be, MO),AP20187 (ARIAD Pharmaceuticals, Cambridge, Mass.) or maturation cocktail(MC), containing 10 ng/ml TNF-alpha, 10 ng/ml IL-1beta, 150 ng/ml IL-6(R&D Systems, Minneapolis, Minn.) and 1 micrograms/ml of PGE2 (CaymanChemicals, Ann Arbor, Mich.). Other methods for differentiating DCs fromPBMC of a patient are described herein (e.g., Examples section).

Lymphoblasts also may be prepared as stimulator cells by activatingpatient lymphocytes, in certain embodiments. Any suitable method may beused to treat lymphocytes and activate lymphoblasts, and an example isprovided herein (e.g., Examples section). Lymphoblasts can be activatedfrom lymphocytes by contacting the latter with one or more suitablestimulants. In certain embodiments, patient lymphocytes are contactedwith one or more suitable interleukins (e.g., IL-2). An amount of aninterleukin often is selected for specific expansion of sensitizedcells, as known in the art (e.g., 60 International Units of recombinanthuman IL-2 can be utilized). Lymphocytes also can be contacted with anagent that interacts with T cells (e.g., binds to a T cell receptor),such as an antibody for example (e.g., OKT3 murine monoclonal IgG2aantibody that binds to CD3 T cell receptor complex). Any suitable mediumcan be utilized for activation of lymphoblasts (e.g., AIM-V medium).

Methods are known in the art for isolating and expanding T cells. Incertain non-limiting T cell isolation and expansion methods,Ficoll-Hypaque density gradient centrifugation can be used to separatePBMC from red blood cells and neutrophils according to establishedprocedures. Cells can be washed with modified AIM-V (i.e., AIM-V(Invitrogen Corporation) with 2 mM glutamine, 10 micrograms/mlgentamicin sulfate, 50 micrograms/ml streptomycin) supplemented with 1%fetal bovine serum (FBS). Enrichment for T cells can be performed bynegative or positive selection with appropriate monoclonal antibodiescoupled to columns or magnetic beads according to standard techniques.An aliquot of cells can be analyzed for cell surface phenotype includingCD4, CD8, CD3 and CD14. Cells can be washed and resuspended at aconcentration of 5×10⁵ cells per ml of AIM-V modified as above andcontaining 5% FBS and 100 U/ml recombinant IL-2 (rIL-2) (in supplementedAIM-V). Where cells are isolated from a HIV+ patient, 25 nM CD4-PE40 (arecombinant protein consisting of the HIV-1-binding CD4 domain linked tothe translocation and ADP-ribosylation domains of Pseudomonas aeruginosaexotoxin A), or other similar recombinant cytotoxic molecule whichselectively hybridizes to HIV, can be added to the cell cultures for theremainder of the cell expansion to selectively remove HIV infected cellsfrom the culture. CD4-PE40 has been shown to inhibit p24 production inHIV-1-infected cell cultures and to selectively kill HIV-1-infectedcells. To stimulate proliferation, OKT3 monoclonal antibody (OrthoDiagnostics) can be added at a concentration of about 10 ng/ml and thecells can be plated in 24 well plates with 0.5 ml per well. The cellscan be cultured at 37° C. in a humidified incubator with 5% CO₂ for 48hours.

In some embodiments, stimulator cells are subjected to a process thatyields inactivated stimulator cells. Inactivated stimulator cells oftenare not capable of dividing, and often are not capable of certainfunctions (e.g., killing other cells). Inactivated stimulator cells arecapable of activating T cells present in the responder cell populationagainst patient antigens. Inactivated stimulator cells often retain cellsurface structure, and generally are capable of presenting antigen toresponder cells (e.g., presentation of antigen by way of MHC to T cellreceptor of a responder cell). Methods for inactivating stimulator cellsare known in the art, which include, without limitation, irradiatingstimulator cells or contacting stimulator cells with mitomycin C.

Combining Stimulator Cells and Responder Cells

Stimulator cells, from a patient or derived from patient cells, andresponder cells, from a donor or derived from donor cells, may becombined with one another to generate activated cytotoxic T cells. Suchactivated cytotoxic T cells generally arise from the responder cellpopulation, and often are “alloreactive,” meaning that they are activeagainst the stimulator cells, which have been inactivated with agentssuch as mitomycin C or by sources of radiation, such a 60-Cobalt or127-Cesium. Without being bound by theory, responder cells include Tcells that are activated by antigens presented by stimulator cells, andthe resulting activated cytotoxic T cells are capable of killing thestimulator cells, and cells of the patient. In certain embodiments,stimulator cells include (i) inactivated dendritic cells differentiatedfrom patient cell monocytes, (ii) inactivated lymphoblasts activatedfrom patient cell lymphocytes, (iii) inactivated patient cell whiteblood cells (e.g., PBMC), and/or (iv) tumor cells, which may or may notbe exposed to interferon to up-regulate HLA antigens. Responder cellsare lymphocytes from a donor in some embodiments. Combining stimulatorcells and responder cells with the expectation of generatingalloreactive cytotoxic T cells sometimes is referred to herein as an“activation reaction” or a one way lymphocyte dendritic cell reaction(LDCR).

In certain embodiments, a donor is selected based on having a partialmismatch of patient antigen information with donor antigen informationand/or a partial mismatch of stimulator information with responderinformation, as described herein. Stimulator cells and responder cellscan be combined in any suitable ratio for generating activated cytotoxicT cells. In certain embodiments, the ratio of stimulator:responder cellscan be from about 1:1 to about 1:20. The stimulator cells and respondercells are combined under conditions conducive to generating activatedcytotoxic T cells. Such conditions can include one or more stimulants(e.g., low dose IL-2 (60 IU/ml for DC stimulator cells). Cultureconditions can include a suitable medium (e.g., AIM-V medium) with orwithout serum (e.g., 5% autologous serum) or heat inactivated andclarified plasma. In embodiments where serum is utilized in culturemedium, cells may be weaned from serum-containing medium over time.Stimulator cells and responder cells may be combined for any suitableperiod of time, including, without limitation, from 2 to 25 or moredays. Responder cells may be re-stimulated one or more times (e.g., 1 to10 or more times) with additional stimulator cells, which can becombined at a stimulator:responder cell ratio described above.Re-stimulation can be for any suitable period of time, such as a periodof time described above for the initial stimulation.

Alloreactive cytotoxic T cells resulting from the combination ofstimulator cells and responder cells can be identified, separated and/orpurified by methods described herein. Cytotoxic T cells also may beadministered to a patient, with or without identification, separation orpurification, to treat a condition or disorder, as addressed in moredetail hereafter.

Characterization of Cells and Activities

Methods for assessing stimulator cells, responder cells and activatedcytotoxic T cells are known in the art. Such methods can be carried outat a suitable time point, and some are performed before patient cellsare exposed to activation or differentiation conditions, beforestimulator cells and responder cells are combined and/or after thelatter cells are combined. For example, certain methods assess theability of antigen presenting cells (e.g., patient cells, DCs,lymphoblasts) to activate responder cells (e.g., donor cells, T cells),and some methods assess the activity of activated responder cells (e.g.,donor cells, T cells). Examples of such methods are described herein(e.g., Examples section).

Presence, absence or amount of cell surface markers and/or production ofcertain cytokines can be utilized to determine whether certain cellshave reached a particular maturation state (e.g., mature dendritic cell,mature and/or activated T cell). Levels of a stimulant in the cytoplasmof cells, or secreted by cells, also can be assessed. For example,activated T cells produce interferon (IFN)γ, which can be assayed asdescribed herein (e.g., using an antibody that binds IFN-γ; Examplessection). Cytokines can be measured in culture supernatants usingcommercially available enzyme-linked immunosorbent assay kits (e.g.,human IL-6 and IL-12p70 (BD Biosciences).

A cell having a certain feature (e.g., one or more cell surface markers)can be identified, separated and/or purified from cells not having thatfeature. Presence, absence of amount of a surface marker facilitatesidentification, separation and/or purification of immunologic cellsknown in the art. For example, cells in a population can be contactedwith an antibody that binds to a particular cell marker on a subset ofthe cells. Cells that display the marker and bind the antibody can beseparated from cells that do not display the marker and do not bind theantibody. A flow cytometer can be utilized to separate certain celltypes from others, and the separated cells can be assessed and/orfurther manipulated.

Cell surface markers expressed, or not expressed, on the cell surface ata particular state of differentiation or activation are known. Forexample, markers are known for cytotoxic activated T cells (e.g., CD8+,CD3+, CD69+); helper T cells (e.g., CD3+, CD4+, and CD8−); T/NK cells(CD3+, CD16+ or CD56+); regulatory T cells (e.g., CD4+/CD25+; productionof certain cytokines (e.g., IL-10 and/or TGF-beta)); helper T cells(e.g., CD4+); human stem cells (e.g., CD34+). DCs express MHC molecules(e.g., HLA class I molecules, HLA class II molecules), co-stimulatorymolecules (e.g., CD80+ (B7.1), CD86+ (B7.2), and CD40+, which areco-receptors in T-cell activation that enhance the DC's ability toactivate T-cells) and chemotactic receptor (e.g., CCR7+). Other markersthat can be detected on DCs include, without limitation, CD11c, CD83 andCD86. DCs may lack markers specific for granulocytes, NK cells, B cells,and T cells. In some instances, DCs express 33D1 (DC from spleen andPeyer's patch, but not skin or thymic medulla), NLDC145 (DC in skin andT-dependent regions of several lymphoid organs and CD11c (CD11c alsoreacts with macrophage)). Agents that bind to markers are known in theart and are commercially available (e.g., antibodies bound to adetectable label) and methods for identifying, separating and purifyingcells using such agents are known (e.g., described herein). Cell surfacestaining can be performed using fluorochrome-conjugated monoclonalantibodies (BD Biosciences, San Diego, Calif.). Cells also can analyzedusing a flow cytometer (e.g., FACSCalibur cytometer (BD Biosciences, SanJose, Calif.)).

Cells can be identified, separated and/or purified before being treated(e.g., differentiation into DCs or activation into lymphoblasts), afterbeing treated, after exposure to a condition that generates inactivatedcells, after being combined with a stimulator or responder counterpart,or after administration to a patient. For example, separated cells maybe exposed to conditions that produce differentiated cells (e.g., DCs),activated cells (e.g., lymphoblasts, activated T cells) and/orinactivated cells (e.g., inactivated DCs, inactivated lymphoblasts), insome embodiments. Separated cells also may be administered to a subjectfor cell therapy (e.g., activated T cells may be administered), incertain embodiments. Separated cells can be substantially free fromother cell types (e.g., substantially isolated). A cell having aparticular marker, or a particular cell type, may represent about 60% ofmore of the cells in a population of cells.

Methods for identification, separation and isolation of cells include,without limitation, flow cytometry (e.g., fluorescent-activated cellsorting (FACS)), column chromatography, panning with magnetic beads,western blots, radiography, electrophoresis, capillary electrophoresis,high performance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, and the like, and variousimmunological methods such as fluid or gel precipitin reactions,immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, and the like.

Labeling agents can be used to label cell antigens, and examples oflabels include, without limitation, monoclonal antibodies, polyclonalantibodies, proteins, or other polymers (e.g., affinity matrices),carbohydrates or lipids, which often are attached, or are capable ofbeing attached, to a detectable label. Detection can proceed by anyknown method, such as immunoblotting, western blot analysis, tracking ofradioactive or bioluminescent markers, capillary electrophoresis, oranother other methods that tracks a molecule based upon size, chargeand/or affinity. The particular label or detectable group used and theparticular assay are not critical aspects of the invention. A detectablemoiety can be any material having a detectable physical or chemicalproperty. Such detectable labels have been well-developed in the fieldof gels, columns, solid substrates cell cytometry and immunoassays and,in general, any label useful in such methods can be applied to thepresent invention. Thus, a label is any composition detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,optical or chemical methods. Useful labels include, without limitation,magnetic beads (e.g. Dynabeads™), fluorescent dyes (e.g., fluoresceinisothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g.,3H, 125I, 35S, 14C, or 32P), enzymes (e.g., LacZ, CAT, horse radishperoxidase, alkaline phosphatase and others, commonly used as detectableenzymes, either as marker gene products or in an ELISA), nucleic acidintercalators (e.g., ethidium bromide) and calorimetric labels such ascolloidal gold or colored glass or plastic (e.g. polystyrene,polypropylene, latex, and the like) beads.

A label can be coupled directly or indirectly to a desired component ofan assay or separation method according to methods known in the art. Asindicated above, a wide variety of labels can be used, with the choiceof label depending on the sensitivity required, ease of conjugation ofthe compound, stability requirements, available instrumentation, anddisposal provisions. Non-radioactive labels often are attached byindirect attachments. A ligand molecule (e.g., biotin) sometimes iscovalently bound to a polymer, in certain embodiments. The ligand thenbinds to an anti-ligand (e.g., streptavidin) molecule which is eitherinherently detectable or covalently bound to a signal system, such as adetectable enzyme, a fluorescent compound, or a chemiluminescentcompound. A number of ligand/anti-ligand pairs can be used. Where aligand has a natural anti-ligand, for example, biotin, thyroxine, andcortisol, it can be used in conjunction with a labeled, anti-ligand, insome embodiments. A haptenic or antigenic compound can be used incombination with an antibody in certain embodiments.

A label can be conjugated directly to a signal generating molecule(e.g., by conjugation with an enzyme or fluorophore) in someembodiments. An enzymes of interest sometimes is utilized as a label,and can be a hydrolase (e.g., phosphatase, esterase, glycosidase), oroxidoreductases (e.g., peroxidases), in certain embodiments. Fluorescentcompounds include fluorescein and its derivatives, rhodamine and itsderivatives, dansyl, umbelliferone, and the like. Chemiluminescentcompounds include luciferin, and 2,3-dihydrophthalazinediones, e.g.,luminol. For a review of various labeling or signal producing systemswhich are used, see, U.S. Pat. No. 4,391,904.

Labels can be detected by methods known in the art. Where a label is aradioactive, for example, a scintillation counter or photographic film(i.e., autoradiography) can be utilized. Where a label is a fluorescentlabel, it is optionally detected by exciting a fluorochrome with theappropriate wavelength of light and detecting the resulting fluorescence(e.g., by microscopy, visual inspection, via photographic film, by theuse of flow cytometers or such-like electronic detectors such as chargecoupled devices (CCDs) or photomultipliers and the like), in someembodiments. Enzymatic labels can be detected by providing appropriatesubstrates for the enzyme and detecting the resulting reaction product,in certain embodiments. Calorimetric labels often are detected simply byobserving the color associated with the label, in some embodiments. Invarious dipstick assays, conjugated gold often appears pink, whilevarious conjugated beads appear the color of the bead, for example.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence ofantibodies. In this case, cells are agglutinated by samples comprisingthe antibodies bound to the cells. In this format, none of thecomponents need be labeled and the presence of the target antibody isdetected by simple visual inspection.

Depending upon the assay or separation technique utilized, variouscomponents, including an antibody, or anti-antibody, sometimes are boundto a solid surface. For instance, in certain embodiments, unwanted cellsare panned out of bone marrow using appropriate antibodies bound to asubstrate over which cells are passed. Methods for immobilizingbiomolecules to a variety of solid surfaces or microbeads are known inthe art. For instance, a solid surface sometimes is a membrane (e.g.,nitrocellulose), a microtiter dish (e.g., PVC, polypropylene, orpolystyrene), a test tube (glass or plastic), a dipstick (e.g. glass,PVC, polypropylene, polystyrene, latex, and the like), a microcentrifugetube, a flask, or a glass, silica, plastic, metallic or polymer bead.The desired component sometimes is covalently bound, or non-covalentlyattached (e.g., through nonspecific bonding) in certain embodiments.Organic and inorganic polymers, natural and synthetic, are known andsometimes employed as a solid surface material. Illustrative polymersinclude polyethylene, polypropylene, poly(4-methylbutene), polystyrene,polymethacrylate, poly(ethylene terephthalate), rayon, nylon, poly(vinylbutyrate), polyvinylidene difluoride (PVDF), silicones,polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, and thelike. Other materials sometimes include paper, glasses, ceramics,metals, metalloids, semiconductive materials, cements and the like.Substances that form gels, such as proteins (e.g., gelatins),lipopolysaccharides, silicates, agarose and polyacrylamides can be used.Polymers which form several aqueous phases, such as dextrans,polyalkylene glycols or surfactants, such as phospholipids, long chain(12-24 carbon atoms) alkyl ammonium salts also can be selected andutilized.

Certain assays can detect cell proliferation. In certain embodiments, Tcells in a responder cell population proliferate in response tostimulator cells, and progress or success (or lack thereof) of anactivation reaction can be assessed. In certain non-limiting examples ofa cell proliferation assay, cells can be pulsed with a radiolabelednucleotide (e.g., tritiated thymidine), and the amount of radiolabelednucleotide incorporated into cellular DNA can be assessed (e.g., thehigher amount of incorporation the high level of proliferation). Anexample of such an assay is described herein (e.g., Examples section).

In some embodiments, certain assays detect one or more ratios ofstimulators (e.g., cytokines) produced during activation reactions. Suchratios can be indicative of the progress or success (or lack thereof) ofan activation reaction. In some assay embodiments, a T helper 1 (Th1) toT helper 2 (Th2) cytokine ratio is assessed. A ratio of suitablestimulators can be assessed, and in some embodiments, a ratio betweenany two of the following stimulators can be determined: IFN-γ, TNF-α,IL-2, IL-4, IL-5 and IL-10. In certain embodiments, a ratio isdetermined for (i) IFN-γ to IL-10, and/or (ii) TNF-α to IL-4.

Certain assays can assess cytotoxic T cell activity by detecting one ormore cytokines generated by activated T cells (e.g.,granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon(IFN)γ, tumor necrosis factor (TNF) α). In a non-limiting example of anIFN-γ assay, DCs from HLA-A2-positive healthy volunteers can be pulsedwith MAGE-3 A2.1 peptide (residues 271-279; FLWGPRALV) on day 4 ofculture, followed by transduction with Ad-iCD40 and stimulation withvarious stimuli on day 5. Autologous T cells can be purified from PBMCby negative selection (Miltenyi Biotec, Auburn, Calif.) and mixed withDCs at DC:T cell ratio 1:3. Cells can be incubated in complete RPMI with20 U/ml hIL-2 (R&D Systems) and 25 micrograms/ml of MAGE 3 A2.1 peptide.T cells can be restimulated at day 7 and assayed at day 14 of culture.For quantification, flat-bottom, 96-well nitrocellulose plates(MultiScreen-HA; Millipore, Bedford, Mass.) can be coated with IFN-γ mAb(2 μg/ml, 1-D1K; Mabtech, Stockholm, Sweden) and incubated overnight at4° C. After washings with PBS containing 0.05% TWEEN 20, plates can beblocked with complete RPMI for 2 h at 37° C. A total of 1×10⁵presensitized CD8+ T effector cells can be added to each well andincubated for 20 h with 25 micrograms/ml peptides. Plates then can bewashed thoroughly with PBS containing 0.05% Tween 20, and anti-IFN-mAb(0.2 μg/ml, 7-B6-1-biotin; Mabtech) can be added to each well. Afterincubation for 2 h at 37° C., plates can be washed and developed withstreptavidin-alkaline phosphatase (1 μg/ml; Mabtech) for 1 h at roomtemperature. After washing, substrate (3-amino-9-ethyl-carbazole;Sigma-Aldrich) can be added and incubated for 5 min. Plate membranesdisplaying dark-pink spots that can be scanned and analyzed by ZellNetConsulting Inc. (Fort Lee, N.J.).

Certain assays for cytotoxic T cell activity can assess the cell-killing(e.g., cell lysis) activity of activated T cells. Certain assays detecta component inside a cell released when it is killed by an activated Tcell, and one example is a chromium release assay. In a non-limitingexample of a chromium release assay, antigen recognition can be assessedusing target cells labeled with 51 Chromium (Amersham) for 1 h at 37° C.and washed three times. Labeled target cells (5000 cells in 50 μA) canbe then added to effector cells (100 μA) at certain effector:target cellratios in V-bottom microwell plates at certain concentrations.Supernatants can be harvested after 6-h incubation at 37° C., andchromium release is measured using MicroBeta Trilux counter(Perkin-Elmer Inc, Torrance Calif.). Assays involving LNCaP cells can berun for 18 hours. The percentage of specific lysis is calculated as:100*[(experimental−spontaneous release)/(maximum−spontaneous release)].

Specificity of activated T cells also can be assessed by methods knownin the art. For example, a tetramer staining assay can be utilized todetermine activated T cell specificity. In a non-limiting example of atetramer staining assay, HLA-A2 tetramers assembled with MAGE-3.A2peptide (FLWGPRALV) can be obtained from Baylor College of MedicineTetramer Core Facility (Houston, Tex.). Presensitized CD8+ T cells in 50μA of PBS containing 0.5% FCS can be stained with PE-labeled tetramerfor 15 min on ice before addition of FITC-CD8 mAb (BD Biosciences).After washing, results can be analyzed by flow cytometry. The assaydescribed in this paragraph utilizes a particular peptide (i.e.,MAGE-3.A2 peptide) that may or may not be applicable to certaintherapeutic methods and compositions described herein, and anotherrelevant peptide may be substituted.

A polarization assay can be utilized to determine whether antigenpresenting cells are capable of activating T cells from a donor byassaying for activated cells that display CD4 and IFN-γ markers. In anon-limiting example of a polarization assay, naïve CD4+CD45RA+ T-cellsfrom HLA-DR11.5-positive donors (genotyped using FASTYPE HLA-DNA SSPtyping kit; BioSynthesis, Lewisville, Tex.) can be isolated by negativeselection using naïve CD4+ T cell isolation kit (Miltenyi Biotec,Auburn, Calif.). T cells can be stimulated with autologous DCs pulsedwith tetanus toxoid (5 FU/ml) and stimulated with various stimuli at astimulator to responder ratio of 1:10. After 7 days, T cells can berestimulated with autologous DCs pulsed with the HLA-DR11.5-restrictedhelper peptide TTp30. Cells can be stained with PE-anti-CD4 Ab (BDBiosciences), fixed and permeabilized using BD Cytofix/Cytoperm kit (BDBiosciences), then stained with hIFN-γ mAb (eBioscience, San Diego,Calif.) and analyzed by flow cytometry. Supernatants can be analyzedusing human TH1/TH2 BD Cytometric Bead Array Flex Set on BD FACSArrayBioanalyzer (BD Biosciences). The assay described in this paragraphutilizes a particular peptide (i.e., peptide TTp30) that may or may notbe applicable to certain therapeutic methods and compositions describedherein, and another relevant peptide may be substituted (e.g., anotherHLA peptide may be utilized and donors having an HLA that presents thepeptide can be selected).

Any suitable assay can be utilized to determine the activity of DCs asthey are differentiated. A migration assay (e.g., chemotaxis assay) canbe utilized to determine whether viable dendritic cells are present in aculture medium, for example, and methods for assessing DC migration areknown in the art. In a non-limiting example, migration of DCs can bemeasured by passage through a polycarbonate filter with 8 micrometerpore size in 96-Multiwell HTS Fluoroblok plates (BD Biosciences). Assaymedium (250 μL) containing 100 ng/ml CCL19 (R&D Systems) or assay mediumalone (as a control for spontaneous migration) can be loaded into alower chamber. DCs (50,000) can be labeled with Green-CMFDA cell tracker(Invitrogen), unstimulated or stimulated for 48 h with the indicatedreagents, and can be added to an upper chamber in a total volume of 50μL for 1 hour at 37° C. and 5% CO₂. Fluorescence of cells, which havemigrated through the microporous membrane, can be measured using theFLUOstar OPTIMA reader (BMG Labtech Inc., Durham, N.C.). The meanfluorescence of spontaneously migrated cells can be subtracted from thetotal number of migrated cells for each condition.

Administration of Cytotoxic T Cells and Treatments

Cytotoxic T cells herein provided may be formulated in a pharmaceuticalcomposition in any manner appropriate for administration to a subject. Acomposition may be prepared by washing cells one or more times with amedium compatible with cells of the subject (e.g., phosphate bufferedsaline). Cells also may be combined with components that form atime-release matrix or gel in some embodiments. Non-limiting examples ofcomponents that form a matrix include, without limitation, fibrin,proteoglycans or polysaccharides. A matrix sometimes is a thrombus orplasma clot in some embodiments.

Compositions comprising cytotoxic T cells can be administered topatients for treatment of a condition. The cytotoxic T cells often areadministered to the same patient, from whom stimulator cells werederived used to generated the T cells. In some embodiments, cytotoxic Tcells are administered to a subject who is not the patient from whichthe stimulator cells used to prepare the T cells were derived.

A composition can be administered to a subject in need thereof in amounteffective to treat a cell proliferative condition (e.g., cancer, tumor),inflammation condition or autoimmune condition. The terms “treat” and“treating” as used herein refer to (i) preventing a disease or conditionfrom occurring (e.g. prophylaxis); (ii) inhibiting the disease orcondition or arresting its development; (iii) relieving the disease orcondition; and/or (iv) ameliorating, alleviating, lessening, andremoving symptoms of the disease or condition. The terms also can referto reducing or stopping a cell proliferation rate (e.g., slowing orhalting tumor growth) or reducing the number of proliferating cancercells (e.g., removing part or all of a tumor).

Given that activated T cells often are alloreactive and can kill cellsof a patient that present patient antigen to which the cytotoxic T cellsare sensitized, the T cells often are administered in a manner that doesnot lead to significant killing of non-afflicted tissue. Activated Tcells also often are administered to a part of the body that does notrapidly inactivate the administered T cells. In certain embodiments,activated T cells can be administered to an immuno-privileged region ofa subject. An immuno-privileged region sometimes is characterized by oneor more of the following non-limiting features: low expression of MHCmolecules; increased expression of surface molecules that inhibitcomplement activation; local production of immunosuppressive cytokinessuch as TGF-beta; presence of neuropeptides; and constitutive expressionof Fas ligand that controls the entry of Fas-expressing lymphoid cells.An immuno-privileged region can be semi-immuno-privileged, where aminority subset of cells are subject to the immune system. In certainembodiments, a composition is administered to the brain, animmuno-privileged region, to treat a cancer, where cancer cells are thepredominant antigen presenting cells and are preferentially killed bythe T cells over non-cancer cells. Other non-limiting examples ofimmuno-privileged regions of the body are portions of the eye (e.g.,ocular anterior chamber, ocular uveal tract, cornea, central nervoussystem), testis, liver and pregnant uterus.

Activated T cells also may be administered to another part of the bodythat is not immuno-privileged, in certain embodiments. In someembodiments, activated T cells are administered to a part of the bodywhere T cells are not substantially cleared or inactivated. For example,activated T cells may be administered directly to a solid tumor mass,where the T cells may not be readily transported to other parts of thebody or inactivated (e.g., injected into the tumor). Compositions can beadministered to the subject at a site of a tumor, in some embodiments.Diffuse cancers are treatable where the composition is maintained incontact with cells within a limited area (e.g., within the cranialcavity), in certain embodiments.

Cytotoxic T cells are delivered in any suitable manner. A dose can beadministered by any suitable method, including, but not limited to,systemic administration, intratumoral administration, bolus injection,infusion, convection enhanced delivery, blood-brain barrier disruption,intracarotid injection, implant delivery (e.g., cytoimplant), andcombinations thereof (e.g., blood-brain barrier disruption followed byintracarotid injection). Blood-brain barrier disruption can include,without limitation, osmotic disruption; use of vasoactive substances(e.g., bradykinin); exposure to high intensity focused ultrasound(HIFU); use of endogenous transport systems, including carrier-mediatedtransporters such as glucose and amino acid carriers, for example;receptor-mediated transcytosis for insulin or transferrin; blocking ofactive efflux transporters such as p-glycoprotein, for example;intracerebral implantation; convection-enhanced distribution; use of aliposome; and combinations of the foregoing. Cytotoxic T cells aredelivered by injection in a suitable volume (e.g., about 5 ml to about20 ml volume (e.g., about 10 ml volume)), and in a suitable medium(e.g., saline; phosphate buffered saline). An implant sometimes includesa gel or matrix. In certain embodiments, an infusion is via a catheterand/or reservoir (e.g., Rickham, Ommaya reservoir).

The dose given is an amount “effective” in bringing about a desiredtherapeutic response (e.g., destruction of cancer cells) by thealloreactive cytotoxic T cells in the composition. For pharmaceuticalcompositions described herein, an effective dose often falls within therange of about 10⁸ to 10¹¹ cells. The cells can include allogeneicstimulators and responders, or may be purified to a certain degree(e.g., substantially pure) for responder cells (e.g., activated Tcells). About 1×10⁹ to about 5×10¹⁰ cells sometimes are delivered, insome embodiments, and in certain embodiments, about 10⁸ to about 10¹⁰cells, about 10⁹ to about 10¹¹ cells, about 10⁸ to about 10⁹ cells,about 10⁹ to about 10¹⁰ cells, about 10¹⁰ to about 10¹¹ cells, about2×10⁹ to about 2×10¹⁰ cells, or about 2×10⁹ to about 2×10¹⁰ cells, aredelivered. Multiple doses can be delivered over time to achieve adesired effect, and often, each dose delivers an effective amount ofcells. Cells in the composition delivered can contain a mixture ofresponder cells and stimulator cells, sometimes in a ratio between about1:1 and about 100:1, and sometimes in a ratio between about 5:1 andabout 25:1, and sometimes about 10:1. In some embodiments, cytotoxic Tcells are purified to a certain degree (e.g., cytotoxic T cells areabout 30% or more of cells in the composition (even more than 95% ofcells in the composition)). Any number of component cells or otherconstituents may be used, as long as the composition is effective as awhole. The number of cells utilized in a composition also can dependculture conditions and other factors during preparation.

A pharmaceutical composition provided herein may be administeredfollowing, preceding, in lieu of, or in combination with, one or moreother therapies relating to generating an immune response or treating acondition in the subject (e.g., cancer). For example, the subject maypreviously or concurrently be treated by chemotherapy, radiationtherapy, surgery, cell therapy and/or a forms of immunotherapy andadoptive transfer. Where such modalities are used, they often areemployed in a way or at a time that does not interfere with theimmunogenicity of compositions described herein. The subject also mayhave been administered another vaccine or other composition to stimulatean immune response. Such alternative compositions may include tumorantigen vaccines, nucleic acid vaccines encoding tumor antigens,anti-idiotype vaccines, and other types of cellular vaccines, includingcytokine-expressing tumor cell lines. Non-limiting examples ofchemotherapeutic agents include, without limitation, alkylating agents(e.g., cisplatin); antimetabolites (e.g., purine, pyrimidine); plantalkaloids and terpenoids (e.g., taxanes); vinca alkaloids andtopoisomerase inhibitors. Surgeries sometimes are tumor removal orcytoreduction, the latter of which is removal of as much tumor aspossible to reduce the number of tumor cells available forproliferation. Surgeries include, without limitation, surgery throughthe nasal cavity (trans-nasal), surgery through the skull base(trans-sphenoidal), and craniotomy (opening of the skull).Radiotherapies include, without limitation, external beam radiotherapy(EBRT or XBRT) or teletherapy, brachytherapy or sealed sourceradiotherapy, systemic radioisotope therapy or unsealed sourceradiotherapy, virtual simulation, 3-dimensional conformal radiotherapy,intensity-modulated radiotherapy, particle therapy and radioisotopetherapy. Conventional external beam radiotherapy (2DXRT) often isdelivered via two-dimensional beams using linear accelerator machines.Stereotactic radiotherapy is a type of external beam radiotherapy thatfocuses high doses of radiation within the body (e.g., cyberknife, gammaknife and Novalis Tx). Cell therapies include, without limitation,administration alone or in combination of dendritic cells, alloreactivecytotoxic T-lymphocytes, stem cells, and monocytes.

A composition may be administered in intervals, and may be replenishedone or more times. A composition may be administered about 1 to about 20times. The time interval between each administration independently maybe of days or even months, for example 1 month to about 6 months, orabout 1 day to about 60 days, or about 1 day to about 7 days. Subsequentadministration of a composition described herein can boost immunologicactivity and therapeutic activity.

Timing for administering compositions is within the judgment of amanaging physician, and depends on the clinical condition of thepatient, the objectives of treatment, and concurrent therapies alsobeing administered, for example. Suitable methods of immunologicalmonitoring include a one-way mixed lymphocyte reaction (MLR) usingpatient lymphoblasts as effectors and tumor cells as target cells. Animmunologic reaction also may manifest by a delayed inflammatoryresponse at an injection site or implantation site. Suitable methods ofmonitoring of a tumor are selected depending on the tumor type andcharacteristics, and may include CT scan, magnetic resonance imaging(MRI), radioscintigraphy with a suitable imaging agent, monitoring ofcirculating tumor marker antigens, and the subject's clinical response.Additional doses may be given, such as on a monthly or weekly basis,until the desired effect is achieved. Thereafter, and particularly whenan immunological or clinical benefit appears to subside, additionalbooster or maintenance doses may be administered.

When multiple compositions are administered to a patient, it is possiblethat an anti-allotype response could manifest. The use of a mixture ofallogeneic cells from a plurality of donors, and the use of differentallogeneic cell populations in each dose, are strategies that can helpminimize the occurrence of an anti-allotype response. During the courseof therapy, a subject sometimes is evaluated on a regular basis forgeneral side effects such as a febrile response. Side effects aremanaged with appropriate supportive clinical care.

In some embodiments, methods and compositions provided herein areutilized to treat a cell proliferative condition. Examples of cellproliferation disorders, include, without limitation, cancers of thecolorectum, breast, lung, liver, pancreas, lymph node, colon, prostate,brain, head and neck, skin, liver, kidney, and heart. Examples ofcancers include hematopoietic neoplastic disorders, which are diseasesinvolving hyperplastic/neoplastic cells of hematopoietic origin (e.g.,arising from myeloid, lymphoid or erythroid lineages, or precursor cellsthereof). The diseases can arise from poorly differentiated acuteleukemias, e.g., erythroblastic leukemia and acute megakaryoblasticleukemia. Additional myeloid disorders include, but are not limited to,acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) andchronic myelogenous leukemia (CML) (reviewed in Vaickus, Crit. Rev. inOncol./Hemotol. 11:267-297 (1991)); lymphoid malignancies include, butare not limited to acute lymphoblastic leukemia (ALL), which includesB-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease. In a particular embodiment, a cell proliferativedisorder is non-endocrine tumor or endocrine tumors. Illustrativeexamples of non-endocrine tumors include but are not limited toadenocarcinomas, acinar cell carcinomas, adenosquamous carcinomas, giantcell tumors, intraductal papillary mucinous neoplasms, mucinouscystadenocarcinomas, pancreatoblastomas, serous cystadenomas, solid andpseudopapillary tumors. An endocrine tumor may be an islet cell tumor.Also included are pancreatic tumors (e.g., as pancreatic ductaladenocarcinomas); lung tumors (e.g., small and large celladenocarcinomas, squamous cell carcinoma, and bronchoalveolarcarcinoma); colon tumors (e.g., epithelial adenocarcinoma, and livermetastases of these tumors); liver tumors (e.g., hepatoma,cholangiocarcinoma); breast tumors (e.g., ductal and lobularadenocarcinoma); gynecologic tumors (e.g., squamous and adenocarcinomaof the uterine cervix, anal uterine and ovarian epithelialadenocaroinoma); prostate tumors (e.g., prostatic adenocarcinoma);bladder tumors (e.g., transitional, squamous cell carcinoma); tumors ofthe reticuloendothelial system (RES) (e.g., B and T cell lymphoma(nodular and diffuse), plasmacytoma and acute and chronic leukemia);skin tumors (e.g., malignant melanoma); and soft tissue tumors (e.g.,soft tissue sarcoma and leiomyosarcoma).

A cell proliferation disorder may be a tumor in an immune-privilegedsite, such as the brain, for example. A brain tumor is an abnormalgrowth of cells within the brain or inside the skull, which can becancerous or non-cancerous (benign). A brain tumor is any intracranialtumor having (and/or arising from) abnormal and uncontrolled celldivision, often in the brain itself (neurons, glial cells (astrocytes,oligodendrocytes, ependymal cells), lymphatic tissue, blood vessels), inthe cranial nerves (myelin-producing Schwann cells), in the brainenvelopes (meninges), skull, pituitary and pineal gland, or spread fromcancers primarily located in other organs (metastatic tumors). Primarybrain tumors sometimes are located infratentorially in the posteriorcranial fossa (often in children) and in the anterior two-thirds of thecerebral hemispheres or supratentorial location (often in adults),although they can affect any part of the brain. Non-limiting types ofbrain tumors include glioma (e.g., mixed glioma), glioblastoma (e.g.,glioblastoma multiforme), astrocytoma (e.g., anaplastic astrocytoma),oligodendroglioma, medulloblastoma, ependymoma, brain stem tumors,primitive neural ectodermal tumor, and pineal region tumors.

As certain embodiments are directed to administering a compositioncontaining cytotoxic T cells can be administered to an immuno-privilegedregion of a subject, any disorder occurring in such a region can betreated. For example, a disorder of the eye, liver, testis or pregnantuterus amenable to treatment by alloreactive cytotoxic T cells can betreated with a composition of cytotoxic T cells described herein.

Certain matters are considered when compositions described herein areutilized to treat a brain tumor. If a tumor mass is resectable or partlyresectable, then the composition can be administered at or near the siteor in a cavity generated by the resection. If a brain tumor iscompletely removed it still often is beneficial to administer thecomposition to surrounding tissue to kill remaining cancer cells. Aconvenient time to administer alloactivated cells to a resectable siteis during the time of surgery, in some embodiments. To keep the cells atthe site until completion of the surgical procedure, it is convenient toadminister the cells in a pharmaceutically compatible artificial gel, orin clotted plasma.

When the solid tumor mass is not resectable, or where less invasiveprocedures are desired, the composition can be injected at or near thetumor site through a needle. For deeper sites, the needle can bepositioned using ultrasound, radioscintigraphy, or some other imagingtechnique, alone or in combination with the use of an appropriate scopeor cannula. For such applications, the cell population is convenientlyadministered when suspended in isotonic saline or a neutral buffer in asuitable volume (e.g., about 5 to about 20 ml (e.g., 10 ml)).

EXAMPLES

The examples set forth below illustrate certain embodiments and do notlimit the invention.

Example 1 Cellular Therapy Studies with Autologous and AllogeneicEffector Cells

MHC can act as a glioma directed antigen. These studies examinelow-passage glioma cell explants by flow cytometry to assess their MHCClass I and II expression. Single cell suspensions were prepared fromdissociated primary brain tumor specimens and placed into culture. Fromthese specimens, tumor cells plated and glia did not.

Fresh normal brain cells were also derived from temporal tip lobectomyspecimens from seizure patients or from autopsy tissue (<8 hr). Braintumor cell explants expressed high levels of MHC Class I antigens(93-100% positive; mean fluorescence intensity or MFI range 4-662), andlittle to no MHC Class II (0.1-7% positive; MFI all below 2 ornegative). Normal brain did not express, or expressed little, Class I(0.5-15%; MFI<2) or II (1-5%; MFI<2.3) antigens. This suggests thatpatient MHC can act as a brain tumor directed antigen in the brain.

Thus, alloCTL directed against tumor-bearing host MHC could be used foradoptive immunotherapy treatment if given intratumorally, since lysis ofcells should be largely restricted to tumor cells while leaving normalbrain cells intact. Other cells in the brain, such as endothelial cells,microglia and reactive astrocytes may express some MHC; however, even ifsome of these accessory cells were injured, they were capable ofrepopulating in the brain.

In vivo animal studies using alloCTL for treatment of rat gliomas.Evidence in a rat model indicated that there was no occurrence ofextreme inflammatory reactions to multiple instillations of alloCTL intonormal brain, and that very focal inflammatory responses result whenthey were placed into tumor-bearing regions of brain. Implantation ofcannulas into the brains of Fischer 344 rats gave us an experimentalmodel in which to study trafficking of adoptively transferred alloCTL incannulated brain. It was found that the alloCTL were capable of movingthrough brain parenchyma, a feature necessary if infiltrating pockets oftumor cells in brain neuropil are to be eliminated. Trafficking ofalloCTL in the caudate-putamen area was shown when placed into brainparenchyma in a site far removed from the cannula and instillation track(right frontal brain). Furthermore, efficacy of alloCTL in eradicatingnew and established intracranial (i.c.) rat brain tumors wasdemonstrated. Repeated i.c. infusions of alloCTL (using either single ormultiple donors) were tolerated well by the animals. Adoptive transferof alloCTL into tumor-bearing animals resulted in all animals exhibitingextended survival compared to sham treated controls and, in addition, acure rate of between 20-35%, which was something not clearly observedwith LAK cells. Despite the immunogenicity documented for the 9L tumormodel, alloCTL-treated animals that succumbed to tumor exhibited anincreased survival relative to sham-treated controls.

In vitro studies using human alloCTL to lyse glioma cells. Human tumorspecimens were often unavailable to use for sensitization oftumor-specific CTL. However, lymphocytes that do express high levels ofHLA can be isolated and expanded from patients. Since brain tumor cellsdisplay HLA class I antigens and normal brain cells do not, HLA can actas tumor-directed antigens if alloCTL are used for brain tumor therapy.

alloCTL are cytolytic toward relevant tumor target cells and demonstratespecificity as determined by cold target inhibition assays and bychromium-release cytotoxicity assays with blocking antibodies to class Imolecules and to the T-cell receptor. With reference to FIG. 7,specificity of alloCTL for relevant glioma target is demonstrated in51Cr-release cytotoxicity assays. CTL specificity is demonstrated byshowing that alloCTL, directed to the HLA antigens of patient 13-06, ismore capable of lysing that patient's glioma cells (13-06-MG), thananother person's glioma cells (DBTRG-05MG) having 2 HLA alleles incommon with 13-06-MG. 51Cr-release assays are the standard fordetermining CTL. It is demonstrated that normal brain cells are nottargets of alloCTL directed toward the HLA antigen of the donor; it isalso demonstrated that nearly 100% of the cells isolated from 18 gliomacell explants displayed HLA class I antigen at variable relative antigendensities. Furthermore, if the display of HLA on gliomas is upregulatedwith exogenous interferon, or if the tumor cells are transduced withvectors coding for the interferon gene, reflective of what may occur ina proinflammatory environment, they are better targets of alloCTL. Inaddition, little change in glioma lysis by alloCTL occurs whenco-incubated in the presence of dexamethasone. Thus, short-term lysis bythe alloCTL is not affected even if the patients treated were onimmunosuppressive steroid therapy.

Generation of alloCTL in artificial capillary systems. alloCTLgeneration in Baxter LifeCell Tissue Culture Bags has been developed asan alternative to the artificial capillary systems (FDA BB-IND 5423reactivation materials, 12/2007). Protocols were developed for the exvivo expansion of stimulator lymphocytes from brain tumor patients usingnon-specific stimulation (OKT3 & IL-2) and growth in artificialcapillary systems. Furthermore, an efficient and cost-effective methodhas been developed to generate responder alloCTL, also in artificialcapillary systems. Responder lymphocytes from healthy unrelated donors,immunologically distinct from the patient, were used as a source ofprecursor alloCTL, and a one-way mixed lymphocyte reaction (MLR)proceeded with irradiated patient lymphocytes as sensitizing cells.

In vitro studies with immunoresistant glioma cell clones. Selectivepressure with multiple alloCTL preparations was applied in vitro to aglioma cell explant derived from a patient at initial diagnosis. Stablyimmunoresistant glioma cell clones were obtained only after theapplication of continuous in vitro selective pressure, but not withintermittent selective pressure. The latter mimicked the in vivotreatment where patients were given alloCTL infusions a month apart overa 10 month period. This suggests that patients do not build up atolerance to repeated intracranial administrations of alloCTL when madefrom single or multiple allodonors.

In summary, it was demonstrated that:

alloCTL precursors (PBMC) directed against an MHC mismatch are readilytransformed into tumoricidal CTL in vitro,

alloCTL placed intracranially are protected long enough in thisimmunologically semi privileged site from a host immune response toperform their effector functions,

alloCTL are capable of trafficking through brain tumor tissue to reachinfiltrating tumor cells,

multiple alloCTL administrations are more efficacious in reducing tumorburden than a single administration,

single or multiple donors of precursor alloCTL can be used for thetherapy of one patient,

repeated, frequent infusions maintain alloCTL presence in the brain (20%of injected dose remained at 1 week),

treatment with ex vivo activated alloCTL may be effective in patients onsteroids to alleviate edema,

patient lymphocytes can be used in place of tumor as the sensitizingcells,

brain tumor cells display high levels of MHC antigen and normal braincells very little,

therapeutic numbers of alloCTL can be produced in artificial capillarysystems (hollow fiber bioreactors) and in tissue culture bags,

there is little likelihood that patients will build up a tolerance tointermittently-applied alloCTL preparations.

Example 2 Clinical Experience

Pilot Phase I Clinical Trial with Intratumorally Administered alloCTL

Patient profiles. Six recurrent glioma patients (ages 26-46 years) weretreated with intracavitary alloCTL and interleukin-2 (IL-2). Withtoxicity as the primary concern, patients with a variety of histologicaltypes were allowed to enroll (see Table 1). The pathologic diagnosesincluded three recurrent glioblastoma multiformes (GBMs), two anaplasticoligodendrogliomas, and one anaplastic astrocytoma. All had failedtreatment consisting of 1 to 3 debulking surgeries and standardradiation (>5000 cGy). All but one also had failed prior chemotherapyand two had additionally failed gamma knife treatment. At entry, thechoices were hospice care or experimental therapy.

TABLE 1 Patients, Immune Therapy, & Status Patient Number/Tumor # ofalloCTL Time to tumor histology cycles progression/SurvivalBTP1/Glioblastoma 2 TTP 3 mos, recurrence at distant site, died at 4 moBTP2/Glioblastoma 2 TTP 3 mos, local tumor recurrence, died at 4 moBTP3/Anaplastic 5 TTP 32 mos, died at 40 mo oligodendrogliomaBTP4/Anaplastic 2 Withdrew from study, alive with oligodendrogliomastable disease at 14 yr BTP5/Anaplastic 5 Completed protocol, live withstable astrocytoma disease at 14 yr BTP6/Glioblastoma 1 TTP < 1 mo, diedat 1 month

alloCTL treatment cycles and clinical status. Five treatment cycles werepossible. Each cycle was given every other month and involved 2 3intracranial infusions of alloCTL within a two week period. Differentdonors were used at each cycle that differed from the patient by 2-3HLA, AB loci (Table 2, matching loci are shaded). All three GBM patientshad tumor recurrence and died before completing 5 cycles. Two otherpatients (BTP3 and BTP5) completed the entire 10-month series. BTP3,with an anaplastic oligodendroglioma, did well until tumor progressionwas noted on MRI at 32 months; he died at 40 months from the start ofimmunotherapy. Patient BTP4, also with an anaplastic oligodendroglioma,experienced side effects during the second treatment cycle and withdrewfrom the protocol. She was still alive, with no evidence of tumorprogression at fourteen years from start of immunotherapy. BTP5, with ananaplastic astrocytoma, was also alive at fourteen years with no signsof tumor recurrence. The three Grade III glioma patients received thehighest number of alloCTL.

Short-term and long-term toxicity. The toxicity and follow-up onpatients treated in this pilot trial were reported. The side-effects ofthe treatment were transient and tolerable (headache, lethargy, feverand nausea). No long-term side effects (as determined by neurologicexams and Karnofsky performance scores) were experienced by thepatients. Although development of GVH was of concern, no signs orsymptoms indicating development of GVH were recorded from the multipleplacements of alloCTL.

alloCTL infusate numbers, phenotypes, and HLA types. The alloCTL numbersand viabilities given to the patients, along with the cumulativephenotypic expression of the CTL infusates were previously summarized.The total lymphocyte doses per patient ranged from 1×10⁸ to 5.2×10⁹.Donors of the precursor alloCTL differed by minimally 2 HLA-AB loci fromthe host. CTL cultures between 12 to 35 days after sensitization wereused for therapy, but in most instances the 2-3 infusions occurredbetween days 14-21. CTL were used as effectors and activated lymphocyteblasts from the patient and donor were used as targets in 4-hr⁵¹Cr-release assays. The percent lysis of the patient targets rangedfrom 29-64% at a 10:1 effector to target (E:T) ratio; whereas lysis ofthe irrelevant donor targets was zero.

TABLE 2 HLA types of patients and donors Patient BTP1 BTP2 BTP3 CYCLE 12 1 2 1 2 3 4 5 HLA type SELF Donor 1 Donor 2 SELF Donor 1 Donor 2 SELFDonor 1 Donor 2 Donor 3 Donor 4 Donor 5 A1 XX X A2 X X X X XX X A3 X X XX A11 X X A24 X A26 X X A28 A29 A31 X A32 A33 X X B7 X X X X X B8 XX X XX B13 X B14 B17 B18 X X B27 B38 B41 X B44 X X B49 B50 B51 X B58 X X B60X X X B62 X B63 X Patient BTP4 BTP5 BTP6 CYCLE 1 2 1 2 3 4 5 1 HLA typeSELF Donor 1 Donor 2 SELF Donor 1 Donor 2 Donor 3 Donor 4 Donor 5 SELFDonor 1 A1 X X X X A2 XX X X X A3 X X X A11 A24 A26 X X A28 X X A29 X XA31 X X X A32 X A33 B7 X XX X B8 X B13 X B14 X B17 X B18 B27 X X B38 XB41 X B44 X X B49 X B50 X B51 X B58 B60 X X B62 XX B63

Neuroimaging and Follow-up. The gadolinium-enhanced MRI of all patients,upon entrance into this study, had to show unequivocal evidence of tumorprogression when measured and compared to a prior scan. Follow-up MRIscans from BTP3, who died at 40 months post-immune therapy wereexamined. Two patients, BTP4 and BTP5, were still alive with stabledisease at 14 years post-immune therapy and routinely receive follow-up;their MRI scans show no change in a series of contrast T1-weighted MRIscans taken.

Tumor bank/HLA-typed tumors. Seventeen low-passage cultured glioma cellexplants and established glioma cell lines that were characterized forHLA and tumor associated antigens (TAA) are provided. For seven of themthe HLA was typed at class I and II alleles by molecular analyses usingRT-PCR SSP (Table 3). For some patients lymphocyte specimens werematched to go along with the tumor.

TABLE 3 HLA-Typed Cultured Glioma Cells 04-11-MG: A*0101; B*08(01, 19N),*5701; Cw*0602, *07(01, 06, 18); DPB1*0101, *0301; DRB1*0301, *1302;DRB3*0101, *0301; DQB1*0201, *0604 D-645MG: A*02(05, 14), 23(01, 02);B*35(01, 42), *4901; Cw*04(01, 09N), *07(01, 06, 18); DRB1*0101,*0405,DRB4*0103; DQB1*0302,*0501 DBTRG05-MG: A*02(01, 04, 09), 68(01, 11N,23); B*35(01, 42), *3801; Cw*12(03, 04), 15(02, 07); DR B1*0402, *14(01,39); DR B3, B4; DQ B1*0302, *0503 NR103: A*0201, B*07(02, 05, 06),*40(01, 33); Cw*0304, *0702; DPB1*0401; DRB1*0408, *1501; DRB4*0103,5*0101; DQB1*0301, *0602 NR106: A*0201; B*1501; Cw*0304; DPB1*1301;DRB1*0405; DRB4*0105; DQB1*0303 NR213: A*0201; B*44(02, 10N, 27), *5501;Cw*0303, *05(01, 03); DPB1*0401, *1101; DRB1*0401, *1501; DRB4*0103,5*(0101); DQB1*0301, *0602 T98G: A*0201; B*3503, *3906; Cw*04(01, 09N),*0702; DPB1*0301,*0401; DRB1*0801, *1201; DRB3*0202; DQB1*0302, *0402

Percentages of CD3+ T cells within the alloCTL preparations displayingactivated markers (CD69+/IFN-γ). Precursor alloCTL were combined withinactivated sensitizing lymphocyte blasts at a responder to stimulator(R:S) ratio of 10:1. At 14 days after the initial one-way MLR, the cellswere restimulated with OKT-3 (10 ng/ml) overnight before analysis usingthe BD Fast Immune Kit. The kit contained surface markers for CD3-APC,CD8-PercP-Cy5.5, CD69-PE and provided for intracellular interferon-γ(IFNγ-FITC) determination. CD69 is an early activation marker and IFN-γgenerally sorts with CD69+ cells.

It was informative to look at those subsets that also expressed CD69 andintracellular IFN-γ. When the activated CD69+ marker was associated witheach of these two subpopulations, they had mean fluorescence intensities(MFI) for IFN-γ that greatly exceeded those MFIs for the individualCD3+/CD4+ (1.5 fold) and CD3+/CD8+ (5.2 fold) subpopulations (see Table4).

TABLE 4 alloCTL Subset Analyses alloCTL Phenotype IFN-γ MFI CD3+ 2503CD3+/CD8+ 1720 CD3+/CD8+/CD69+ 8950 CD3+/CD4+ 2600 CD3+/CD4+/CD69+ 3792

Response by the alloCTL CD8+ subset to incubation with relevant target.An example is provided in Table 5 from a different alloCTL preparation.The subset of CD3+/CD8+ cells that were also CD69+ was 10.5%, and halfof that subset (i.e., 52.1%) also expressed IFN-γ at a MFI equal to 500.When that same alloCTL preparation was incubated with relevant gliomatargets for 18 hr, the percentage of CD3+/CD8+/CD69+ cells went up6-fold. A third of those cells also expressed IFN-γ at a 5-fold higherMFI. When alloCTL from the same preparation were analyzed forCD3+/CD4+/CD69+ cells, upon incubation with relevant target thepercentage rose 2.3-fold, but less than 1% of them were IFN-γ positive.Thus, the CD3+/CD8+ cells react by producing proinflammatory IFN-γ uponexposure to the relevant HLA glioma antigens.

Flow cytometric analyses are quite sensitive and can detect phenotypicsubsets present in small percentages. The CTL precursor frequency tomajor antigen can be as high as 10%. With approximately 7-14 doublingspossible over a 2 to 3 week alloCTL culture period, enrichment of thealloCTL effectors responding to restimulation should be detectable inthe alloCTL pool. Examining the fold increases in the activated CD3/CD69subsets producing IFN-γ upon exposure to relevant target cells isproposed.

7-AAD flow cytometric assays determine cell injury caused by alloCTL totargets displaying relevant HLA antigen(s). During cell injury, theplasma membrane becomes increasingly permeable and a fluorescent DNAdye, 7-AAD that selectively binds to guanosine/cytosine regions of theDNA, is taken up by the cells in proportion to the degree of injury.Scattergrams are generated from this flow cytometric-based assay thatdistinguish live, early apoptotic, and dead/late apoptotic cells.

The images in FIG. 8 show carboxyfluorescein diacetate succinimidylester-labeled human 13-06-MG glioma cells that were or were notco-incubated with anti-13-06 alloCTL for 4 hours at an E:T ratio of10:1. By the 7-AAD assay, the percentages of cell injured (apoptotic anddead) totaled 74% from a starting population that was 85% viable.Significant glioma cell injury occurred within a very short time.

Significant glioma cell injury occurred quite rapidly upon theircoincubation with alloCTL. This assay appears to be an alternative assayto the chromium release cytotoxicity assay. Apoptotic/necroticsegregation was confirmed, demonstrating that cells within the “deadregion” were positive for propidium iodide, and ≧75% of the cells withinthe “apoptotic region” stained with annexin-V that binds the earlyapoptotic marker, phosphatidylserine.

TABLE 5 Phenotypic analysis of activated T-cell subsets within alloCTLbefore/after exposure to relevant target glioma. % of CD3+ alloCTL +/−T-cell subset % of CD3+ cells subset also IFN-γ relevant glioma targetphenotype with phenotype IFN-γ+ MFI alloCTL CD3+/CD8+/CD69+ 10.5% 52.1%500 alloCTL + target glioma CD3+/CD8+/CD69+ 62.7% 34.7% 2543  alloCTLCD3+/CD4+/CD69+ 35.3%  0.7% 254 alloCTL + target glioma CD3+/CD4+/CD69+  80%  0.9% 1605 

alloCTL can induce glioma cell apoptosis and lysis. In an vitromorphologic assay of hematoxylin and eosin (H&E) stained cells, gliomacells were exposed to alloCTL directed against the HLA antigens presenton the glioma. Additional evidence of the significant cell injurycapability of the alloCTL to glioma cells in a short period is visibleafter only a 4-hr exposure of the alloCTL to the monolayer of gliomacells.

In addition to obvious lysis of glioma cells from the monolayer culture,apoptotic cells are also revealed by their condensed or fragmentednuclei. Vesiculation of cells is also visually apparent. H & E stainingmorphologically demonstrates apoptotic cells glioma cells exposed toalloCTL. 13-06-MG glioma cells cultured for 4 hours in the absences ofalloCTL show normal, non-apoptotic brain tumor cells, which were largein size, contained ample cytoplasm, and had large oval nuclei. 13-06-MGglioma cells co-incubated with anti-13-06-MG alloCTL demonstrate classicmorphologic changes: condensed nuclei, fragmented nuclei, apoptoticbodies, and membrane blebbing. There was evidence of the DNAfragmentation of a large glioma cell (CellTracker Orange labeled) causedby T effector cells (CellTracker Green labeled) in close conjunction tothe glioma cell. Also, the quick, recycling capability of cytolytic Tcells was shown. CTL can rapidly lyse tumor cells. Three tumor cells incontact with one effector cell were lysed in 480 sec. One CTL effectorcell staining positive for granzymes was shown in contact with 3 tumorcells. CTL had the ability to “recycle” and lyse more than one tumorcell in a short period when they came into contact with them. In each ofthe successive panels, one tumor cell bound to the CTL was lysed. Over aspan of 480 sec, one CTL bound to three tumor cells induced the lysis ofall three targets

Example 3 Dendritic Cells

Dendritic Cell Studies. PBMC recovered from melanoma patients weresubjected to density gradient centrifugation. The enriched adherentmonocyte fraction was cultivated in serum-free AIM-V medium supplementedwith rhGM-CSF and rhIL-4 for 6 days. The adherent monocytic cells can bevisualized with projections by inverted light microscopy. Inverted lightphotomicrographs can also depict human monocyte derived, immature DC(based on low CD83 expression) displaying typical membrane projectionsafter one week in vitro cultivation.

Flow cytometry revealed that these DCs were 98% positive for HLA-DRclass II and these DCs were also characterized for other costimulatorymolecules (see Table 6). In vitro monocyte-derived DCs werecharacterized by flow cytometry using four color flow cytometry. Thethird decade expression (MFI) of HLA-DR, CD11c, CD80, CD83, and CD86surface expression on the DC was noted. They may be classified asimmature based on their lower level CD83+ surface expression, but theyhad high CD11c, CD86, and CD80 expressions.

DCs were capable of macropinocytic function by incubation withdextran-FITC for 60 min at 4° C. and at 37° C. Active uptake was alsodemonstrated. DCs were tested for their functional ability to uptakefluorescently-labeled dextran. Following removal of 0 minute samples,dextran-FITC was added to DCs. Cells were incubated 60 min at 4° C. or37° C. After incubation, cells were washed, fixed, and analyzed by flowcytometry. The results supported the active uptake by DC(macropinocytosis).

TABLE 6 DC expression of standard surface markers Surface MarkersPercent Positive HLA-DR 98 CD11c 98 CD80 20-25 CD83 <1% CD86 60-70

Dendritic cells are strong activators of the allogeneic lymphocyticresponse because of their high surface level expression of HLA class Iand II and a number of other minor histocompatibility antigen molecules.In order to investigate this functional allogeneic stimulatorycapability, standard immature DCs were setup in a one-waylymphocyte-irradiated dendritic cell reaction (LDCR) and the resultingallogeneic lymphocytes were tested for proliferative response. Resultsshow lymphocytes proliferate in response to incubation with irradiatedDCs, whereas they do not when incubated with uncultivated monocytes. Theproliferative response of allogeneic T cells to DCs. Mean cpm±SD werecalculated from three triplicate wells. Enriched, uncultivated monocyteswere frozen, stored, and used as control stimulator cells.

It was also demonstrated that DC-lymphocytes interact in vitro after a 4hr co-incubation. Multiple lymphocytes are also seen in contact witheach other. Elongated adherent DCs interacting with small lymphocyteshave been observed in vitro. Bifurcation of the DC's terminal end, whereit and the lymphocyte are contacting one another, also was noted. Alsoseen were large adherent DC with projections wrapping around smallrefractile lymphocytes.

Example 4 Structural Analysis by an Algorithm

Analysis of mismatched HLA eplets by an algorithm: brain tumor patientsand their alloCTL donors used in the pilot clinical study. Brain tumorpatients (BTP) 3, 4 and 5 treated in the pilot clinical trial exhibitedprolonged survival after alloCTL immunotherapy. The patients and donorswere serologically HLA typed. Each healthy donor differed by at leasttwo HLA AB loci from the patient. Serological HLA types of the patientsand healthy donors were converted to the most likely molecular HLA typesbased on race/ethnicity. FIG. 1 shows the molecular HLA types of BTP3, 4and 5, and the HLA types of the donor's whose alloCTL they receivedduring therapy. To determine the eplets that were mismatched betweeneach patient and their alloCTL donors, the presumed molecular HLA typesof the patient:donor pairs were then entered into the algorithm programand the number of mismatched eplets was quantified for eachpatient:donor pair. Not surprisingly, the three responding BTP generallyreceived higher cumulative numbers of mismatched eplets (range 42-117)during their course of treatment, compared to the nonresponders (range20-59), but this likely related to the responding patients receivingmore infusions. More interestingly, it was found that certain epletmismatches were exclusive to the responding BTPs. The 151RV and 62QEwere mismatched in at least one treatment for each responding patient(FIG. 1), while no mismatches were found to be exclusive to patients whodid not respond. Additionally, it is noteworthy that several other epletmismatches were associated with the responder group, being present inmultiple alloCTL preparations administered to the patients (i.e. 9T,56R, 166DG, etc). In contrast, there were no mismatched eplets common tothe multiple alloCTL preparations administered to nonrespondingpatients.

The significance of the preclinical work in this study is that it canlead to an improved and consistent alloCTL in vitro generation methodresulting in potent cytotoxic alloreactive killers. As well, the workcan lead to a better personalized allodonor selection for gliomapatients in the cellular therapy trial; this would be based upon ananalysis of the molecular HLA types of the responding donor lymphocytesrelative to that of the patient.

Example 5 Comparison of alloCTL Molecular/Cellular and FunctionalCharacteristics when Generated Using One-Way Mixed Lymphocyte Reaction(MLR) Versus Those Made by One-Way Lymphocyte Dendritic Cell (DC)Reaction (LDCR)

The ability to differentiate human monocytes in vitro into DC usingrecombinant growth factors is a new opportunity to use DC as stimulatorsfor optimizing the in vitro generation of alloCTL used for the cellulartherapy of brain gliomas. Molecular/cellular and functionalcharacteristics of alloCTL currently generated by a standard 1-way MLRtechnique were compared to those when generated by a technique employingstimulation by 1-way LDCR.

An alternative method of using DC presentation of alloantigen optimizesthe generation of potent cytolytic alloreactive killers and inducesproinflammatory responses. It is expected that alloCTL generated by LDCRhave stronger lytic activity to relevant HLA-bearing targets than thosegenerated by the standard 1-way MLR technique. Activated, mature DCdisplay very high levels of HLA molecules compared to other cell typesincluding lymphoblasts; they are also strong antigen-presenting cells(APC). The exploitation of the high level of surface HLA expression byDC in generating alloCTL results in consistent alloCTL generation withstrong lytic alloreactive activity.

Peripheral blood mononuclear cell (PBMC) populations from differentindividuals as responders and stimulators were used. For one-way MLR,irradiated stimulator lymphoblasts are mixed with responder lymphocytesfrom normal, healthy HLA-mismatched donors. For one-way LDCR, themonocytes were differentiated into stimulator DC first. Irradiated,activated, and mature DC were mixed with responder lymphocytes fromhealthy, HLA-mismatched donors to produce alloCTL. The alloCTL generatedby each technique was tested for their molecular, cellular andfunctional characteristics to evaluate their respective alloreactivity.

In particular, FIG. 5 shows that therapeutic alloCTL are generated whenirradiated lymphoblasts isolated from a brain tumor patient are mixedwith PBMC isolated from a healthy donor in a one-way MLR. AlloCTL werecultured in medium with low concentration IL-2. Cytolytic function andcytokine production by cells of appropriate phenotype were assayed onday 14 post MLR.

For alloCTL preparations generated from the same responder/stimulatorpairs by MLR or by LDCR, the following was performed:

Determination of the cytotoxicity of the alloCTL to relevant target,i.e., stimulator lymphoblasts displaying the HLA to which they aresensitized.

Determination of the fold-increase in the phenotypic subset displayingthe activated T cell marker (CD3/CD69) that produces IFN-γ within thealloCTL upon exposure to relevant target, i.e., stimulator patientlymphoblasts displaying the HLA to which they are sensitized.

Determination of the proliferative response of the alloCTL upon exposureto relevant target.

Determination of the soluble Th1 to Th2 cytokine ratios (i.e., IFN-γ toIL-10 or TNFα to IL-4) produced upon alloCTL exposure to relevanttarget.

Methods for Example 5

Sources of responder and stimulator cells. To obtain PBMC forpreclinical studies: (1) normal blood donor collections at 100 ml orless, (2) purchase of buffy coats from the San Diego Blood Bank, and (3)limited leukapheresis of donors were performed. Donors had to testnegative for all infectious disease agents. The density gradientisolated PBMC was washed then fractionated, using standard plasticadherence, into monocytes and lymphocytes. The nonadherent cells fromthe PBMC containing T, B and NK cells was either used fresh orcryopreserved in vials containing 107-108 cells for the MLR generationmethod. From experience using PBMC as responders, the MLR could beapplied equally well to fresh PBMC as well as to vitally-frozen PBMC.For LDCR, the adherent monocytes was differentiated to DC.

Standardizing alloCTL generated by one-way MLR or LDCR. Irradiatedstimulator (S) lymphocytes and responder (R) lymphocytes were employedfrom normal, healthy HLA-mismatched donors. The strategy was to use asmall pool of young (18-50 years old) normal blood donors to helpstandardize the PBMC reactivity to alloantigen. PBMC from older peopledid not respond to antigenic stimulation as well, i.e., they hadquantitative and functional defects in the CD4 T helper cell compartmentand cells that lack CD40L. Furthermore, it was demonstrated that restinglymphocytes, activated lymphocytes (aka lymphoblasts), as well aslymphocytes or lymphoblasts that have been cryopreserved and thenthawed, all have high HLA surface expression levels, thus couldadequately serve as stimulators.

Isolation and expansion of stimulator lymphocytes for sensitization ofalloCTL by MLR. A 100 ml blood draw yielded 1 2×108 PBMC after isolationfrom Ficoll density gradients. After washing several times with Hank'sbalanced salt solution (HBSS), the PBMC was suspended in 20 ml of AIM Vsynthetic medium containing 5% autologous serum. The cells were injectedinto the extracapillary space (ECS) of the artificial capillarycartridge and perfused with medium containing Orthoclone OKT3 antibody(50 ng/5×107 cells) and 240 IU/ml of rIL 2. The perfusion volume wasdoubled every 2 4 days by adding fresh rIL 2 containing medium. Lacticacid concentration was measured daily (7 days/week, YSI Statlactate/glucose analyzer) to determine the rate of lactate production(usually 0.2 0.25 gm/109 cells/day). Cells were fed every 4 to 5 days orwhen the concentration of lactate was at 0.5 0.7 gm/liter. Lactic acidproduction paralleled the expansion rate of the cells.

Multiple vials of stimulator lymphocytes were vitally-frozen so therewas the capability of performing multiple alloCTL cultures from anygiven responder to stimulator (R:S) pairs; minimally 3 cultures weregenerated from one R:S pair. The number of stimulator PBMC frozen wasbased upon starting cultures at a R:S ratio of 10:1. Cells harvestedfrom one starter culture were cryopreserved in 10% DMSO/autologous serumand stored at 80° C. The stimulator lymphocytes were thawed prior toinactivation with gamma-irradiation (127Cs-source, 2000 Rads), thenwashed before combining with allogeneic responder lymphocytes.

Isolation of monocytes and generation of stimulator DC. PBMC isolatedfrom whole blood by density gradient centrifugation was washed 2× withHank's balanced salt solution (HBSS). The PBMC was suspended at adensity of 5×106/ml in serum-free, AIM V synthetic medium in plastictissue culture flasks.

After 30 min incubation at 37° C., the nonadherent cells containinglymphocytes were recovered and cryopreserved; the adherent monocyticcells were washed with HBSS to removed loosely adherent cells thenoverlaid with fresh AIM-V medium and cultivated overnight at standardconditions. The next day the adherent cells were washed with HBSS toremove residual platelets, then overlaid with AIM-V medium supplementedwith 1,000 IU/ml of GM-CSF and 500 IU/ml of rIL-4 and cultivated for 6days to differentiate monocytes into immature DCs. At day 6, the mediumwas supplemented with recombinant human TNF-α, IL-6 and IL-1β (10 ng/mlfor each cytokine) and cultured an additional 2 days to mature the DCs.Other studies support an estimate that approximately 10% of the startingcell number are obtained as mature DCs. The DCs were subjected togamma-irradiation (127Cs-source, 2000 Rads), and washed 1× with HBSS inpreparation for the LDCR protocol; these represent the stimulator DCs.

Generation of alloCTL by one-way MLR. Responder PBMC, from a donorgenetically distinct from the donor supplying the stimulator cells, wasisolated with Ficoll Hypaque and washed 2× with HBSS. The responderlymphocytes was combined with 127Cs-irradiated stimulator lymphocytes,at a responder to stimulator (R:S) ratio of 10:1 (i.e., one-way MLR).They were placed into the artificial capillary cartridges and cultivatedat 37° C. with 5% CO₂ with AIM V medium containing 5% autologous serumand 60 International Units (IU)/ml of rIL 2 for 14 days; the cells overa 7 10 day period were weaned from serum containing medium. Arestimulation of the alloCTL occurs at day 12 post-MLR with relevantlymphoblasts at a R:S of 10:1 [48]. Cytotoxicity assessments,proliferation, phenotypically-defined cytotoxic subsets and cytokineproduction were determined on day 14 post-MLR cells as described inlater methods.

Generation of alloCTL by one-way LDCR. The allodonors used forresponders or pCTL were HLA-disparate to the donor supplying stimulatorcells. The adherent cells were grown with growth factors that encourageDC (immature) growth. Growth factors were then to be added to theculture medium to mature the DC.

Briefly, the plastic adherent monocytic cells were cultured in serumfree AIM-V medium supplemented with 1000 units/ml rhGM-CSF and 500units/ml rhIL-4 at 37° C. in a humidified, 5% CO2 incubator. Six dayslater, the immature DC were stimulated with recombinant human TNF-α,IL-6 and IL-1β (10 ng/ml for each cytokine) to induce their maturationfor 2 days. DCs were harvested, irradiated and combined with responderPBMC for LDCR at a R:S ratio of 10:1. The DC presented alloantigen(i.e., stimulators) to the T lymphocytes of the allodonor in thepresence of low dose IL-2 (60 IU/ml). Reactive responder lymphocytesdeveloped into alloCTL capable of recognizing the HLA on the stimulatorcells over a 12 day period. They were restimulated with DC at a 10:1 R:Son day 12 post-LDCR and assessed 2 days later in 4 hour 51Cr-releasecytotoxicity assays, for proliferation, and for phenotype and cytokineproduction.

Example 6 Methods Specific to Example 5A-D Methods Specific to Example5A

Chromium release cytotoxicity assays. alloCTL preparations weregenerated from the same R:S pairs by either MLR or by LDCR. It wasdetermined whether the cytotoxicity of the alloCTL to relevant target,i.e., stimulator lymphoblasts displaying the HLA to which they weresensitized. 51Cr-release assays can be used to determine the lyticactivity of alloCTL effector cells when they were co-incubated with thetarget cells.

Four hr assays were run in 96-well plates at multiple effector to target(E:T) ratios of 3:1, 10:1, 30:1 with triplicate samples as previouslydescribed in other publications. Percent specific release was calculatedby the formula: [(cpm experimental−cpmspontaneous)/(cpmmaximal−cpmspontaneous)]×100%. Spontaneous release wasmeasured for targets in assay medium alone and maximal release were beproduced by lysis of the targets with 2% Triton X-100 (Sigma, St. Louis,Mo.). Lysis obtained at each given E:T ratio was determined and thethresholds of low, moderate and high cytotoxicity can be definedaccordingly.

Day 14 alloCTL generated by 1-way MLR and 1-way LDCR were compared.Statistical assessment of lytic activity and the effects of reactiontype (MLR vs LDCR), the three E:T ratios evaluated as an ordered factor,the three samples, and their possible interactions were made by 2×3×3ANOVA with planned post-hoc comparisons. All statistical operations forthis and all subsequent methods are accomplished in R, version 2.9 orhigher. Optimization of alloCTL by DC presentation was consideredpossible if the cytotoxic responses, by DC-generated alloCTL compared to1-way MLR generated alloCTL, against stimulator lymphoblast target cellswas >15% higher when all data were grouped and normalized from threeequivalent E:T ratios tested.

The alloCTL preparations should have the ability to elicitalloantigen-specific immune responses against relevant target cells invitro. PHA-stimulated lymphoblasts can be used as target cells, whichdisplay high levels of HLA antigen. “Relevant” targets were thelymphoblasts derived from stimulator PBMC. Responding donor lymphoblastsexpress HLA that should be regarded as “self” and therefore should notbe targets of the alloreactive T cells but as a background, negativecontrol. Additionally, K562 natural killer (NK)-sensitive cell targetsdid not express HLA antigen and could be used as “irrelevant” targetcells to assess nonspecific injury caused by NK cells(non-MHC-restricted killing) that was unrelated to T-cell alloreactivity(MHC-restricted killing).

Lysis of K562 was subtracted from stimulator lymphoblast lysis for thesecomparisons also. The levels of HLA expression by lymphoblasts wasanalyzed by flow cytometry using the pan HLA-ABC antibody (W6/32) toassess whether the cytotoxicity directly relates to the relative antigendensity (MFIs) of HLA on the relevant target cells.

Exemplary Variation. A variation of the above described example isillustrated in FIG. 6, which shows the percentage lysis from51Cr-release 4 hr assays at 3 E:T ratios (black, 20:1; red, 10:1; blue,5:1). alloCTL were made with 5 different R:S pairs in MLRs, and thenumbers on the abscissa refer to the one-way MLR number. Statisticalsignificance was evaluated using two-way ANOVA and Bonferroni post-tests(*p<0.05). These data were representative of two separate experimentsperformed with triplicate wells.

Methods Specific to Example 5B

Phenotypic characterization of activated, mature dendritic cells.Aliquots of DC were stained with monoclonal antibodies (mAbs) against DCsurface markers (anti-HLA class I conjugated to fluoresceinisothiocyanate (FITC), anti-HLA class II DR conjugated to PerCp,anti-CD11c conjugated to APC, anti-CD80, anti-CD83, and anti-CD86conjugated to phycoerythrin (PE) (BD Biosciences/Pharmingen, San Diego,Calif.) on ice for 1 hour. The cells were washed three times with coldPBS before analyzing on an LSR II flow cytometer.

Phenotypic characterization of activated, CD3 cytotoxic T cell subsetsby flow cytometric analyses. The cytotoxic subsets with alloCTLpreparations were utilized for production of IFN-γ, because thisparticular cytokine has previously been shown to be most relevant to theTh1 cell-mediated responses to immunotherapy exhibited by T lymphocytes.Additionally, IFN-γ has been used as an in vitro monitoring tool topredict GVH in renal transplant patients where slight mismatches indonor to patient HLA were expected.

With alloCTL preparations generated from the same responder/stimulatorpairs by MLR or by LDCR, the fold-increase in the phenotypically-definedCD3/CD8 cytotoxic subset displaying the activated T cell marker (CD69)that produces IFN-γ within the alloCTL upon exposure to relevant target,i.e., stimulator patient lymphoblasts displaying the HLA to which theywere sensitized, was determined. The cell subset positive for CD3, CD8,CD69, and intracellular IFN-γ (BD Fast Immune Kit, BD Biosciences) wasdetermined at 24 hr after incubation with or without relevant targetcells (stimulator lymphoblasts at a R:S of 10:1). In the last 6 hr ofthe 24 hr incubation, 10 μg/ml of Brefeldin A, a secretion inhibitor,was added. Nonstimulated or stimulated alloCTL were each aliquoted intothree tubes (106 cells/tube) and pelleted at 100× g. Flow cytometricanalysis was performed, staining for cell surface markers (e.g., CD3+,CD8+, CD69+) and cytoplasmic IFN-γ cytokine expression. The Fix and Permreagents e used where indicated according to the manufacturer'sprotocol. In brief, alloCTL cell pellets were resuspended and incubatedwith a fluorochrome-conjugated monoclonal antibody (mAb) cocktail on icefor 30 minutes. The cells were washed, fixed and permeabilized, thenincubated with a fluorochrome-conjugated mAb specific for IFN-γ for 30min. Following the second antibody incubation, the cells were washedagain and resuspended in PBS and analyzed by flow cytometry. Theanalyses were performed with a six-color capable BD LSR II flowcytometer. Percentages of the positive activated T cell subset and themean fluorescence intensities (MFI) of IFN-γ were obtained.

The fold increases in the percentages of the activated subset in thealloCTL that were restimulated versus those not were determined. Aswell, the fold increases in the MFIs for IFN-γ in the alloCTL subsetsthat were or were not restimulated were determined. Each of thesemeasures can be useful for prediction of the extent of cytolysis. It wasnoted that an increase in the cytotoxic subset or the degree of IFN-γexpression that was >1.5-fold may reach significance based upon other'sobservations with patient PBMC in vaccine trials for glioma. Helping tovalidate this approach, others showed that data collected by this flowcytometric method compare well to that collected by limiting dilutionanalyses and supports use of this methodology for subset analysis.

Methods Specific to Example 5C.

Determination of the proliferative response of the alloCTL made by MLRor DC presentation upon their exposure to relevant stimulatorlymphoblasts. The CTL precursor frequency within a donor mononuclearcell pool to patient HLA antigens was variable. That may be as high as10% to allogeneic MHC antigen or as low as 0.1-0.01%. Anticipating thatthe precursor CTL frequency was identical in any givenresponder/stimulator pair, it was possible to determine in theexperiments here if DC presentation was better than T lymphocytepresentation in an MLR in enhancing the proliferative events ofalloresponders. The overall intent was to generate therapeuticallysignificant quantities of alloCTL. The ability of T cells to proliferatewhen exposed to the antigens that they were sensitized to has been usedas an indicator of the presence of antigen-specific CD4+ helper T cells.

The proliferative response of the alloCTL preparations was characterizedupon their exposure to relevant patient lymphoblasts displaying the HLAto which they were sensitized. With alloCTL preparations generated fromthe same R:S pairs made by MLR or by LDCR, the proliferative response ofthe alloCTL was determined upon their exposure to relevant stimulatorlymphoblasts and convert them to stimulation indices for comparison.

The capacity to proliferate in response to HLA presentation by relevantstimulator cells were measured by tritiated thymidine uptake at a R:Sratio of 10:1. In response to the alloCTL seeing relevant antigen,proliferation should ensue. After 48 hr, the culture was pulse-labeledwith 3H-thymidine. DNA synthesis, as a measure of proliferation, wasquantified by using a liquid scintillation counter to measure the amountof radiolabeled thymidine incorporated into the DNA. A stimulation index(SI) was calculated by dividing the number of cpm for the resensitizedalloCTL by the number of cpm for the cells incubated without sensitizingcells.

The SIs obtained for each alloCTL preparation was categorized as havinga high proliferative population versus a low proliferative population.The in vitro proliferative capacity of the alloCTL was compared to theircytotoxicity (see Example 5A), phenotypic analyses (Example 5B), and thelevel of HLA mismatch between the responder and stimulator (Example 7).In general, while there is some consensus in the literature thatproliferative events correlate with responder/stimulator MHC disparitiesat Class II, while cytolytic activity is a function of disparities atClass I, it is possible to confirm the separation of proliferative andcytolytic functions by analyzing the data with molecular HLA types ofthe responder and stimulator. This was addressed using both conventionaland robust regression analyses. In addition to comparing theproliferative differences in alloCTL generated by MLR vs LDCR methods,it was also possible to discern if proliferation of the alloresponderenriched cultures at restimulation resulted from HLA Class IIdisparities, whereas the functionality of the cells as determined bycell injury, and cytotoxic cell phenotype/cytokine production, relatedto HLA Class I disparities between responder and stimulators.

Methods Specific to Example 5D

Determination of the soluble Th1 to Th2 cytokine ratios produced uponalloCTL exposure to relevant target. Other researchers have comparedIFN-γ/IL-10 ratios as an in vitro monitoring tool for assessing tumorhost response using PBMC pre- and post-vaccination, and for T cellinduced GVH development and rejection in transplant patients. WithalloCTL preparations generated from the same R:S pairs by MLR or byLDCR, it is possible to determine the soluble Th1 to Th2 (i.e., IFN-γ toIL-10 or TNF-α to IL-4) cytokine ratios produced upon alloCTL exposureto relevant stimulator target. It is observed that higher Th1 to Th2ratios were correlated with induction of a proinflammatory response invivo and/or correspond to better cytolysis to relevant target.

Supernatants from alloCTL coincubated for 24 hr in the presence orabsence of relevant irradiated stimulator lymphoblasts were examined.The cell suspensions were clarified by refrigerated centrifugation at400×g for 10 min. The clarified medium, or dilutions of it if necessary,were analyzed using the BD cytometric bead array system. The cytokinesto be tested include Th1 and Th2 cytokines IL-2, IL-4, IL-5, IL-10,gamma interferon (γ-IFN) and tumor necrosis factor alpha (TNF-α). Thearray system allowed for collection of multiple cytokine results from asingle small sample at relatively sensitive levels of detection (2.0-4.0pg/ml). Therefore, the processes not only analyzed IFN-γ/IL-10 ratiosbut other alternative Th1/Th2 cytokine permutations (i.e., TNF-α/IL-4)as well. For this reason, the array was a cost effective alternative toELISAs specific for the four cytokines was considered.

Statistical Evaluation. Statistical analysis was performed by abiostatistician. For statistical analysis in Example 5, the data wasdescribed using conventional and statistically robust techniques. Datadescriptions include standard 5-point summaries as well as the firstfour moments and MAD (median absolute deviations). To elucidate theinterrelationships of functional alloresponsiveness (i.e., cytotoxicityof the alloCTL, fold-increases in the phenotypic subset displaying theactivated T cell marker, proliferation in response to exposure torelevant antigens and/or proinflammatory cytokine production) relativeto HLA mismatch, correlative studies included both pairwise analyseswith confidence intervals and additional analyses to investigatesystematic nonlinearities. Both conventional ANOVA and its robustanalogues were used to investigate the relationships. It was possible tocompare the mean averages of triplicate samples in three separateexperiments using the same R:S pairs of at least 15 alloCTL preparationsmade by both methods. The number of experiments needed depend upon thepilot data and power analyses. The implication to obtainingsignificantly higher cytotoxic assessments with alloCTL generated byLDCR vs MLR was an alteration of the generation of alloCTL for clinicalstudies in the existing IND to the FDA.

Interpretation of Data and Alternative Approaches. Generally, withalloCTL preparations one might assume that the proliferative events andcytolytic activity correlate with responder/stimulator HLA disparitiesat Class II and Class I, respectively. This could be reconfirmed usingHLA molecularly-typed individuals for HLA-A,B,C and HLA-DR,DQ allelicdifferences.

At present, alloCTL populations made by MLR for the clinical trial mustmeet three minimal release criteria before they can be administered.First, the gross phenotypes of all alloCTL populations must show thatthe preparations were >60% CD3+. Second, the viabilities of thecultures, determined by trypan blue dye exclusion counts on ahemocytometer, must be >60%. Third, the minimum cytotoxicity they mustexhibit to patient lymphoblasts is 30% lysis at a 30:1 effector totarget ratio (E:T). It was expected that alloCTL made by LDCR wouldconsistently meet those minimum requirements. If the presently proposedLDCR were inadequate, it was quite possible that the activation state ofthe DCs could dramatically influence alloCTL generation and function. Itwas possible to explore agents such as ssRNA, dsRNA, LPS, imiquiod, orother toll-like receptor (TLR) agonists that would affect the TLRexpressivity by the DC and then measure alloreactivity by the alloCTLpreparations.

A 51 Cr-release cytotoxicity assays can be substituted with the 7-aminoactinomycin D (7AAD) flow cytometric based cytotoxicity assay as anonradioactive, rapid alternative, albeit the latter was a cell hungrytechnique. For proliferation, a nonradioactive alternative to tritiatedthymidine was the BrdU Flow Kit (BD Biosciences). That kit could provideBrdU and 7-AAD staining along with surface phenotype, such as CD3, topermit the enumeration and characterization of cells that were activelysynthesizing DNA (BrdU incorporation) in terms of their cell cycleposition (i.e., G0/1, S, or G2/M phases as defined by 7-AAD stainingintensities).

Example 7 The Algorithm Evaluates and Predicts Suitable HLA PartialMismatches Between Alloresponder and Stimulator Lymphocyte Pairs thatWill Induce the Generation of Potent Cytotoxic Alloreactive Killers

It was determined whether the extent or type of HLA eplet mismatchbetween responder and stimulator lymphocytes correlated with (1) the invitro ability of the responding donor alloCTL to lyse stimulatorlymphoblasts; or upon alloCTL exposure to relevant target cells, would(2) cause increases in the appearance of cytotoxic subsets producingproinflammatory IFN-γ cytokine, or (3) cause a skew to higher Th1 to Th2secreted cytokine ratios. It was noted that nonpermissive HLA mismatchbetween the responder and stimulator (either number and/or specific typeof immunogenic eplets) as recognized by the HLAMatchmaker (HLAMm)algorithm modified for cellular response were predictive of functionalalloreactivity.

HLAMm is an algorithm configured to provide structurally based HLAmatching. In particular, when HLAMm is applied to the diverse HLArepertoire, it is able to reliably predict B cell driven alloantibodygeneration following organ transplantation. HLAMm operates by findingpermissible mismatch between molecularly HLA-type donors and recipientssuch to minimize rejection.

Two phases were employed for this work. Example 7A involves a“Discovery” phase where in vitro functional assessments (i.e., cytolysisof relevant target, appearance of phenotypic cytotoxic subsets orproinflammatory cytokine induction to target exposure) were used todrive the modification of the cellular version of the HLAMm program.Versions of the HLAMm computer program can determine quantitativeestimates of structural compatibility and identification of specificeplets or other amino acid configurations (some that are alreadyassociated with T cell induced GVH disease) that might have relevancehere, since theoretically a strong alloresponse is desired. Example 7Binvolved a “Validation” phase where matched glioma patient lymphocytesand glial tumor specimens were available as stimulator cells andrelevant tumor targets, respectively, along with prospectively chosenallodonors from a pool of HLA-typed individuals that HLAMm wouldcategorize as robust or nonrobust allodonors. The modified HLAMm programwas validated for successful in vitro prediction of alloCTL functionalalloreactivity.

A training set using molecularly HLA-typed responder and stimulatorlymphocytes for the HLA Matchmaker (HLAMm) cellular program was created.It was also determined whether the extent or type of HLA eplet mismatchbetween the stimulator cells and the responder donor PBMC was indicativeof the in vitro ability of the donor alloCTL to lyse stimulatorlymphoblasts, or upon exposure to relevant HLA target antigen, tosignificantly increase the activated T cell subset (CD3+/CD8+/CD69+)producing the proinflammatory cytokine, IFN-γ, or to skew the secretedcytokines to a higher Th1/Th2 ratio (i.e., IFN-γ:IL-10 or TNF-α/IL-4).

HLAMm, used in the transplantation setting to predict alloantibodyresponses, is based upon analysis of mismatched epitopes defined byso-called eplets that are configurations of polymorphic amino acidresidues within a 3-Angstrom radius. The computer program appliesdifferent algorithms to consider the two major causes of HLAmismatch-induced bone marrow (BM) transplant patient mortality:engraftment failure and graft-versus host (GVH) disease. HLAmismatch-induced engraftment failures occur during the earlypost-transplant period and appear to involve antibody-mediatedmechanisms, whereas HLA mismatch-induced GVH disease is primarilyinduced by alloreactive T cells, which interact through their T cellreceptors (TCR) with alloepitopes on mismatched HLA molecules. Thecurrent algorithm has accurately predicted alloantibody responses intransplant patients according to the number of structurally definedmismatched epitopes of the donor, and it does not as reliably predictthe T cell induced GVH disease.

Structurally-based HLA matching at the amino acid level by HLAMm forhematopoietic stem cell transplantation was inaccurately hypothesized tobenefit transplant patient survival. Permissible matching at the aminoacid level had only a modest effect on engraftment and acute GVHdisease, and it did not benefit patient survival. Interestingly,structural mismatching at the intermediate level seemed to convey thehighest risk for acute GVH disease; this finding was consistent with CTLprecursor data reported by the Leiden transplant group. Other algorithmswere structurally based and include the polymorphism of thepeptide-binding groove and the structural aspects of the T cell receptor(TCR)—HLA contact area. Obviously, where permissive mismatches wererequired for transplant patients, nonpermissive mismatches wereidentified that would lead to good cytotoxic allokillers. Understandingof the structural and functional basis of T cell alloreactivity wasuseful to ultimately enable choosing donors that provide functionallyrobust alloCTL based on HLA eplet mismatch between patient and donor.HLAMm findings using best-fit serologic HLA data from earlier pilotclinical trial with alloCTL indicated that the three responders, butnone of the non-responders, had mismatches at two eplets, 151RV and62QE.

The degree of cytotoxicity to stimulator lymphoblast target cells byeach alloCTL preparation made from HLA-typed R:S pairs was assessed. Itwas noted that the alloCTL preparations with higher percentages of lysisto relevant target correlate with specific HLA mismatch eplets. Foldincreases in each alloCTL preparation were assessed for the activated Tcell subset with IFN-γ expression upon exposure to relevant stimulatorlymphoblasts. It was noted that alloCTL preparations with significantfold increases (i.e., >1.5-fold) in the activated cytotoxic T cellsubset producing IFN-γ upon exposure to relevant target correlate withspecific HLA mismatch eplets. Production of Th1 and Th2 cytokines wasdetermined by BD Cytometric Array and ratios of IFN-γ/IL-10 andTNF-γ/IL-4 in the supernates of alloCTL coincubated with irradiatedstimulator lymphoblasts were assessed. It was noted that secretedTh1/Th2 ratios that were >1:1 (proinflammatory) correlate with specificHLA mismatch eplets between responder and stimulator pairs.

Methods for Example 7

AlloCTL Generation and Determination of Alloresponsive Functionality.Either of the methods in Example 5 can be used for alloCTL preparationin this example. Cytotoxicity assays, phenotypic analyses, and cytokinedeterminations and their statistical evaluations were as described inExample 5.

Cytotoxicity of Relevant Target. Relationships were drawn between thedegree/type of HLA mismatch (structural) of responder/stimulator pairsand the alloresponsive characteristics (functional) of each alloCTLpreparation. In particular, lysis of target lymphoblasts by the alloCTLin 4 hr assays (triplicate samples at effector:target ratios of 3:1,10:1, and 30:1) was evaluated and normalized. Normalized cell lysis bythe alloCTL of stimulator lymphoblasts are categorized as low (e.g.,achieving between 0-33% cell injury), moderate (say, between 34-65% cellinjury), or high (>66% cell injury). Both conventional regressionmodeling with the general linear model and its robust analogues wereused to assess the HLA mismatch and alloresponsive characteristicsacross effector:target ratios. In vitro cytotoxicity data is correlatedwith the HLA mismatch structural evaluations.

Appearance of Cytotoxic T Cell Subsets Producing ProinflammatoryCytokine Upon Exposure to Relevant Targets. The phenotypic analysis ofthe alloCTL preparations by flow cytometry using the BD Fast Immune Kitallowed a determination of the CD3+/CD8+/CD69+/IFN-γ+ fold increasesachieved upon restimulation of the alloCTL with irradiated stimulatorlymphoblasts. The fold increases is specifically related to mismatchedHLA-eplets (number and type). For purposes of multi-factorial analysis,fold increases of the cytotoxic T cell subset producing proinflammatorycytokine were analyzed by categorizing each alloCTL preparation into oneof three cytotoxic T cell categories. Although subject to modificationonce the data were collected, definitions for categories were used asfollows: (1) low as <1-fold increase in CD3+/CD8+/CD69+/IFNγ+, (2)intermediate as >1 but <1.5-fold CD3+/CD8+/CD69+/IFNγ+, and (3) highas >1.5 CD3+/CD8+/CD69+/IFN γ+. Because the alloCTL were labeled withCFSE before incubation with stimulator lymphoblasts, it was possible todistinguish between R and S cells. Additionally, since it was possibleto analyze the T cell subset that was CD3+/CD8+, the T-helper cell CD4+phenotype was analyzed by default as cells that were CD3+/CD8−. ThreeCD4+ helper/inducer T-cell categories were similarly defined as low,intermediate, and high. The fold increases obtained in the activated Tcell subsets within alloCTL preparations, and the CD4:CD8 ratios thatwere also CD69+, were correlated with HLA mismatches and analyzed bypolytomous logistic regression analyses.

Production of Th1/Th2 Cytokines Upon Exposure to Relevant Targets.Supernates from alloCTL coincubated for 24 hr in the presence ofirradiated stimulator lymphoblasts were examined for cytokine secretionas described earlier. The Th1 and Th2 cytokines assessed by BDcytometric bead array included IL-2, IL-4, IL-5, IL-10, IFN-γ and TNF-αusing clarified medium (100 μA aliquots). While ratios of four cytokines(IFN-γ, IL-10, TNF-α, IL-4) can be measured using sandwich ELISA kits atlevels of detection of 2.0-4.0 pg/ml, the array system was a costeffective, time-saving alternative. Again, subject to modification basedupon the data collected, ratios of Th1:Th2 cytokines >2.0 were used thatwere considered to be highly proinflammatory, ratios <2.0 but >1.0 wasproinflammatory, and ratios <1.0 were anti-inflammatory. Otherpermutations of the Th1 to Th2 cytokines were assessed, such asIL-12/IL-10, or additive Th1 and Th2 cytokine ratios (i.e.,IFN-γ+TNF-α/IL-10+IL-4) were analyzed if dichotomous results wereobtained. IL-2 was disregarded as it was a component of the medium inwhich the alloCTL was maintained and thus likely to be at aconcentration in a nonlinear range. These analyses were chosen as ratiosof the Th1- to Th2-type cytokine producing cells (i.e., IFNγ/IL-10ratios) because they were informative before in predicting rejection, aT-cell driven response in transplant patients, and as a response inimmunotherapy treated (vaccinated) patients.

HLA Typing. The responders and stimulators were HLA typed by highresolution molecular DNA methods for class I HLA-A,B,C and class IIHLA-DR,DP,DQ using RT-PCR sequence specific primers (SSP) or by sequencebased typing (SBT). Serological typing may also accompany theseanalyses. As in Example 5, lymphocytes from young, normal donors wereused.

Serum screening methods for HLA antigens include complement-dependentlymphocytotoxicity (CDC) determined by direct testing, such as the NIHstandard and Amos modified tests, and anti-human globulin augmentation(AHG) technique and in antigen-binding assays such as flow cytometry,ELISA, and Luminex.

It should be noted that high and intermediate resolution methods may beemployed. In general, a “high resolution method” is defined as anymethod that results in specific sequence data, while an “intermediateresolution method” is defined as a method that provides at least partialsequence data. Examples of high and intermediate resolution methodsinclude sequence based typing (SBT), sequence specific primer (SSP),restriction fragment length polymorphism (RFLP), or sequence specificoligonucleotide (SSO) methods.

Such high or intermediate resolution methods also include molecular DNAsequencing methods, molecular RNA sequencing methods, and molecularprotein sequencing methods and may be employed to determine patient cellinformation and/or donor cell antigen information.

HLAMatchmaker (HLAMm) Algorithm. The class I and class II HLAMatchmakerprograms used for transplant rejection predictions are posted at thewebsite: http://www.hlamatchmaker.net/. Here it was tested whether theextent/type of HLA mismatch at the epitope level between the stimulator(i.e., clinical translation=brain tumor patient) and the responder(i.e., clinical translation=alloCTL donor) was predictive of thefunctional properties of the alloCTL. The number and type of mismatchedeplets (for functional epitopes) were analyzed between responder andstimulator at HLA class I and class II alleles by HLAMm. It wasdetermined whether a significant relationship existed between the invitro-collected functional data sets and the ability of the cytotoxicvariant of the HLAMatchmaker program to predict the robustness of thefunctional response. The program's parameters can be modified to bereflective of the in vitro data involving cytolysis and Th1 responses.

Initially, a data set was collected (e.g., as in Example 7A) andutilized as a training set for the algorithm. Validation of the HLAMmalgorithm occurs in Example 7B. The resulting HLAMm algorithm provides auseful tool in clinical studies if it allows a prediction offunctionally active allCTL and selection of appropriately mismatcheddonors for brain tumor patients based on molecular HLA types.

Characterization of structural mismatches. Direct alloreactivity wasseen when T cells restricted to one HLA molecule were exposed to APCbearing a different, but related HLA molecule. Many of the contactsinvolved in TCR antigen recognition involve binding of TCR elements tothe HLA antigen-presenting face, and because of allelic structuraldifferences, the binding of the stimulator alloHLA to a TCR on aresponder may be with greater affinity than to cognate self-HLA. Aswell, aa substitutions in the antigen binding groove of HLA couldcontribute to alloreactivity. Therefore, it was possible to focus on twoaspects: (1) those HLA residues on the TCR “docking” face which wereexposed and capable of taking part in direct TCR binding, and (2) thoseHLA residues lining the HLA antigen-binding groove, which are capable ofparticipating in antigen binding. Since the enhanced affinity of the TCRfor alloHLA+ peptide may result from (a) changes in TCR/HLAinteractions, (b) changes in TCR/peptide interactions, or (c) acombination of these, comparator algorithms were developed to look atthese two classes of allelic changes first separately, then incombination, seeking predictive correlations between structuraldifferences and allostimulation potential. A “mismatch scoring”procedure was developed for related HLA alleles that was predictive ofalloresponses associated with the mismatch.

Two conditions were used to evaluate structural based matching tocellular immune alloresponsive functionality (see Table 7). First, threegeneral mismatch levels involving direct contact between HLA and TCRwere considered. Group 1 related to structurally identical or verysimilar mismatch between alleles of responder and stimulator that mighthave low alloresponsive functionality. Group 2 related to mismatcheswith an “intermediate” level of structural mismatching would cause morecytolysis of relevant target cells or increase the cytotoxic subset uponrestimulation. Group 3 related to structurally very dissimilarmismatches would have less responsiveness than Group 2 because theself-restricted T-cell repertoire would have a lower alloreactivepotential for such mismatches. The multivariate analysis suggested acorrelation between grades II to IV acute GVH disease and increasing lownumbers of triplet/patch mismatches (from 0 to 4), but the 5+triplet/patch mismatches had actually a lower incidence of GVH disease.Moreover, another transplant group reported lower cytotoxic T cellprecursor frequencies towards more structurally divergent mismatches.

Second, the polymorphisms of the peptide-binding groove were consideredbecause they affect the HLA bound peptide repertoire recognized by Tcells. Mismatched alleles with differences in groove residues will binddifferent repertoires of peptides including the minor histocompatibilityantigens and these variations depend on the number of residuesubstitutions and key residues in the binding pockets. While the bindingpocket polymorphisms could be described with patches similar to eplets,previously reported concepts about binding patterns such as thesupertypes described by Sidney et al. BMC Immunology 9 (2008) 1 and byDoytchinova et al. J Immunol 172 (2004) 4314-23 were applied.

TABLE 7 Predicted Alloresponse Based Upon HLA Allele Mismatch of R and SR:S Mismatch- R:S Mismatch- Net Predicted Combination Allele 1 Allele 2Alloreactive Response 1 Low Low Very Low 2 Low Intermediate High 3 LowHigh Low 4 Intermediate Intermediate Very High 5 Intermediate High High6 High High Low to Very Low

For each responder/stimulator allele mismatch, the two corresponding HLAstructures were compared. A “mismatch scoring” algorithm was developedto quantify the structural mismatch between pairs as low, intermediateand high. Recalling that alloreactivity was the result of T cellsencountering a foreign HLA molecule(s) that was sufficiently similar toself to allow TCR/HLA interactions, yet sufficiently different totrigger the activation of a subset of those cells, it was noted thatintermediate level mismatching may result in the strongest alloreactiveresponse. Assuming that to be the case, and further assuming additivealloresponses across two mismatched alleles, Table 8 shows predicted netalloreactive responses from different HLA mismatch combinations.

A characterization of structural mismatch with the alloreactive responsebetween stimulator and allo-responder involves the number/type ofeplets, with binding avidity to the docking interface of the TCR/HLA, orto polymorphic patches in the peptide binding groove. It was possible toobserve and record other eplets with amino acid configurations that wereassociated with functional alloresponsiveness. Functional assessmentsincludes the alloCTL preparation's cytotoxicity to HLA-relevant targetcells, or upon alloCTL exposure to relevant stimulator targets, theappearance of a higher percentage of cells displaying the cytotoxicphenotype that was producing IFN-γ, and a skew to proinflammatorycytokine secretions. Three sets of in vitro data (cytotoxicity,cytotoxic phenotype, cytokine secretion) were analyzed relative to thevarious HLA structural mismatch approaches. The combined information wasanalyzed with conventional and robust multivariate regression analysesto determine systematic underlying relationships across approaches.

The multifaceted approach that was taken incorporates current conceptsfor the in vitro collected information. In addition to HLAMm, otherinvestigators have used serologic crossreactive group (CREG) typing, orstructural approaches such as Histocheck that applies the so-calleddistance index of Risler to assess functional similarities between aasubstitutions on disparate HLA molecules, or other counting of aaaccording to physiochemical properties, but these efforts have largelybeen unsuccessful in the transplantation field. Other issues consideredincluded alloreactive-enriched preparations that could also containnatural killer (NK) cells. Although the alloCTL preparations wereenriched for allokillers, the NK cell impact on the cytotoxicityobtained was considered; data collected using non-MHC expressing,NK-sensitive K562 target cells in the cytotoxicity assays; and thateffect subtracted. The structural matching approach includedpolymorphisms in the 77-82 sequence positions that could be consideredas sites for NK cell inhibition or activation along with the associatedKIR polymorphisms.

The structural basis of indirect allorecognition raised the possibilityof a single allele mismatch generating immunogenic allopeptides thatcould be presented by the matched alleles of the HLA phenotype. Severalinvestigators have developed computer algorithms that predict for anyprotein, the most likely nonamer peptides that could bind to a givenclass I allele(http://www.imtech.res.in/raghava/mmbpred/algorithm.html). This programcould be readily used for mismatched alleles.

The HLAMm was modified to predict functional in vitro cytolytic and Th1responses to glioma target cells by alloCTL made with particularresponder:stimulator pairs. With matched patient glioma and lymphocytes,it was possible to determine the cytotoxicity to patient glioma whenpatient HLA-expressing lymphoblasts were used for stimulation of thealloCTL. This developed a tool that allowed selection of allodonors ofspecific molecular HLA types that predicted robust/nonrobust alloCTLcytolytic or proinflammatory response to the relevant tumor targetcells.

The ability of the modified HLAMm algorithm to prospectively predictfunctional in vitro cytolytic and Th1 responses to glioma target cellsby alloCTL made with particular responder:stimulator pairs was tested.It was noted that slighter degrees of HLA mismatch (number and/or typeof eplets) between donors and patient, as recognized by the modifiedHLAMatchmaker algorithm, would result in more cytolytic allokillers thanthose HLA mismatches that were totally disparate. Alternatively, it waspossible to find that the extent of overall eplet mismatch was not asimportant as certain types of mismatches that might consistently appearin multiple donor mismatches. The information gathered was useful as ascreening method for choosing donors whose PBMC will consistentlygenerate robust alloresponses to the patient's HLA antigens present onthe glial tumor but not on normal brain glia. The clinical extrapolationwas that molecular HLA analyses between allodonors to the glioma patientwere predictive of either acute toxicity and/or response to treatment inpatients treated with intratumoral alloCTL.

Methods in Example 7B

Matching criteria established during research were applied to a separatecohort of data coming from matched glioma patient lymphocyte and tumorspecimens in a tumor bank. The modified HLAMm program was used topredict stimulator:responder pairs that would result in robust alloCTLfunctionality. Cytotoxicity, phenotype, and cytokine response followingco-incubation with target cells were measured as above in Example 7A.

Validation Using Patient Tumor/Lymphocyte Sets as Targets and Stimulatorof alloCTL.

A tissue bank contains matched patient lymphocyte and glioma specimens.Tissue in the bank was collected using an IRB-approved protocol, underwhich patient confidentiality was maintained. The patient lymphocytesacted as stimulators of alloCTL. The cultured glioma specimens acted asrelevant targets in cytotoxicity assays. It was documented that tumorcells in situ and in culture express MHC class I antigens.

It was possible to use precursor alloCTL from a pool of molecularly-HLAtyped, young 18-50 year old allodonors to maximize the likelihood of avigorous response. The specificity, effector function, and avidity ofalloCTL were predicted by the modified HLAMm program, based on the HLAtype of the alloCTL precursor (responder) and stimulator cells.Stimulator:responder pairs could be chosen that the program wouldpredict to be robust or nonrobust based upon molecular differences inHLA type. For this, it was possible to obtain PBMC from glioma patientsand close relatives expected to have little HLA variability, and aswell, PBMC from unrelated donors of different race/ethnicity expected tohave large HLA variability.

The validation phase was performed with several patients matchedtumor:lymphocyte sets (i.e., tumor targets:stimulator lymphocytes) andseveral allodonors as responders for each of the tumor:lymphocyte sets.The ability of HLAMm to predict function was assessed, especiallycytotoxicity using multiple alloCTL preparations from the variousallodonors. The validation models employed both conventional and robustlinear regression and analysis of variance and covariance models forcontinuous data.

When choosing extensively mismatched donor PBMC this will generatenonrobust alloCTL responses, whereas minimizing the extent of HLAmismatch might generate the most efficacious alloCTL responses, i.e.,minor HLA mismatch may better signal aberrant self and induce a betterallo reaction than total mismatch. If it was found that no correlationexists between the extent of HLA mismatch and the degree of alloCTLresponsiveness, these data would suggest that other factors might beplaying a critical role in the generation of cytotoxic alloCTL. Otherparameters to consider induce evaluating apoptosis induction relative tolysis by 7AAD assays instead of Chromium release cytotoxicity assays.There was evidence that effector CTL caused direct cell injury uponcontact with glioma cells by perforin/granzyme-mediated lysis and byinduction of tumor cell apoptosis. It was also possible that findingother combinations of Th1/Th2 cytokine secretions relative to HLAdifferentials to feed into the HLAMm algorithm also was betterpredictive.

It was noted that patient treatment by intratumoral adoptive transfer ofalloCTL may include not only the effects of adoptively transferred, exvivo activated alloCTL, a passive immunotherapy component, but inaddition, an active immunotherapy component as well. Microglia and otherAPCs could engulf apoptotic glioma cells, which were then capable ofpresenting endogenous tumor-associated antigens to circulating Tlymphocytes. This process of cross-presentation has the overall effectof increasing the patient's CTL precursor frequency and function againstTAA. Similar to what has been previously observed in tumor vaccinationprotocols, it was expected that increases in endogenous tumor-specificanti-glioma CTL activity following adoptive transfer of alloCTL. Ifobserved, these increases would suggest that the endogenous immunesystem (active immune therapy) played a role in a cellular (passivetherapy) approach.

This was easily tested by using both patient tumor and patientlymphoblasts as targets. After accruing patients to a dose escalationclinical study involving repeated intratumoral placements of alloCTL inrecurrent glioma patients, it was then determined whether HLAMmprediction of the number and type of mismatched HLA-eplets could also berelated to the acute toxicity the patient may experience after infusionwith a particular alloCTL infusate. In addition, that same informationgathered over the entire treatment period for any patient's alloCTLrepertoire could be related to patients segregated intoresponder/nonresponder groups.

An increase was expected in the frequency of host CD8+“cytotoxic” Tcells following alloCTL immunotherapy, presumably responding to TAAs onthe host glioma cells damaged by adoptively transferred alloCTL, aftermultiple cycles of treatment. However, it was possible that the CTLpfrequency to tumor antigen would be very low in glioma patients. Afterfirst subtracting “self with no TAA” values from “self with TAA” values,the differences between pre- and post-treatment CD8+ cytotoxic T-cellactivity could be assessed. This was useful insight as to the HLAdifferences required of donors to yield cytotoxic alloreactive killers.This information enhanced the response rate to brain tumor adoptivecellular immunotherapy with alloCTL.

Final Considerations

While the invention has been described in connection with the abovedescribed embodiments, it is not intended to limit the scope of theinvention to the particular forms set forth, but on the contrary, it isintended to cover such alternatives, modifications, and equivalents asmay be included within the scope of the invention. Further, the scope ofthe present invention fully encompasses other embodiments that maybecome obvious to those skilled in the art and the scope of the presentinvention is limited only by the appended claims.

1. A method of preparing alloreactive cytotoxic T cells comprising:providing patient cell information, wherein the patient cell informationcomprises patient cell antigen information at least partly determinedthrough one or more of serotyping or a high or intermediate resolutionmolecular sequencing method of major histocompatibility complex (MHC)information; generating stimulator information from the patient cellantigen information; comparing the stimulator information to responderinformation generated from donor cell antigen information, wherein thedonor cell information is determined at least partly through a high orintermediate resolution molecular sequencing method of MHC information;identifying presence or absence of a partial mismatch between thestimulator information and the responder information among patient celland donor cell pairs; selecting a patient cell and donor cell pair toprepare alloreactive cytotoxic T cells based on the presence of thepartial mismatch; and combining cells of the patient with cells of thedonor in an alloreactive cytotoxic T cell reaction based on the patientcell and donor cell pair.
 2. The method of claim 1, wherein the patientcell antigen information is determined at least partly through a high orintermediate resolution molecular sequencing method of MHC information.3. The method of claim 1, wherein the patient cell antigen informationis determined at least partly through a high or intermediate resolutionmolecular DNA sequencing method.
 4. The method of claim 1, wherein thepatient cell antigen information is determined at least partly through ahigh or intermediate resolution molecular RNA sequencing method.
 5. Themethod of claim 1, wherein the patient cell antigen information isdetermined at least partly through a high or intermediate resolutionmolecular protein sequencing method.
 6. The method of claim 1, whereinthe donor cell antigen information is determined at least partly througha high or intermediate resolution molecular DNA sequencing method. 7.The method of claim 1, wherein the donor cell antigen information isdetermined at least partly through a high or intermediate resolutionmolecular protein sequencing method.
 8. The method of claim 1, whereinthe high or intermediate resolution molecular sequencing methodcomprises one or more of a sequence based typing (SBT), sequencespecific primer (SSP), restriction fragment length polymorphism (RFLP),or sequence specific oligonucleotide (SSO) method.
 9. The method ofclaim 1, wherein providing patient cell information comprises providingpatient cell information derived from HLA class I or HLA class IIantigen information; and wherein the donor cell information isdetermined at least partly through a high or intermediate resolutionmolecular sequencing method of HLA class I or HLA class II antigeninformation of a responder and a stimulator.
 10. The method of claim 1,wherein providing patient cell information comprises providing patientcell antigen information derived from HLA class I antigen information;and wherein the donor cell information is determined at least partlythrough a high or intermediate resolution molecular sequencing method ofHLA class I antigen-type information.
 11. The method of claim 10,wherein the cell antigen information is derived from HLA class I alphahelix, beta helix, or peptide binding groove.
 12. The method of claim 1,wherein providing patient cell information comprises providing patientcell antigen information derived from HLA II antigen information; andwherein the donor cell information is determined through a high orintermediate resolution molecular DNA method of HLA class IIantigen-type information.
 13. The method of claim 1, wherein identifyingthe presence or absence of a partial mismatch comprises identifyingeplet partial mismatch information.
 14. The method of claim 1, whereineither or both of the responder information or the stimulatorinformation are generated from one or more of monocytes, antigenpresenting cells, dendritic cells, lymphocytes, lymphoblasts, or Tcells.
 15. The method of claim 1, wherein identifying the presence orabsence of a partial mismatch comprises employing a computer algorithmto identify the presence or absence of the partial mismatch.
 16. Themethod of claim 1, wherein the computer algorithm is configured toprovide structurally based HLA information to identify amino acidmismatches.
 17. The method of claim 15, further comprising the step oftraining the algorithm with a training set of data.
 18. The method ofclaim 17, wherein one or more of cytotoxic T cell activation orcytotoxic T cell activity are compiled in the training set.
 19. Themethod of claim 1, further comprising the step of exposing the cells ofthe patient to conditions generating inactivated cells of the patient.20. The method of claim 19, further comprising the step of detectingpresence or absence of cytoxic T cell activation.
 21. The method ofclaim 1, further comprising the step of administering the alloreactive Tcells to the patient.
 22. The method of claim 21, wherein administeringthe alloreactive T cells to the patient comprises administering thecells to the patient having a cancer.
 23. The method of claim 22,wherein the cancer is a brain cancer.
 24. The method of claim 22,wherein the cancer is a brain glioma, a brain stem glioma, or aleptomeningeal gliomatosus of carcinomatosus.
 25. The method of claim21, wherein the allorective T cells are administered to animmune-privileged site.